Chimeric molecules providing targeted costimulation for adoptive cell therapy

ABSTRACT

The present invention relates to a chimeric molecule useful in adoptive cell therapy (ACT), and cells comprising the same. The chimeric molecule can act as a modulator of cellular activity enhancing responses when an endogenous T-cell receptor (TCR) is engaged with its cognate antigen. The present invention also provides proteins, nucleic acids encoding the chimeric molecule and therapeutic uses thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS FOR PRIORITY

This application is a continuation of U.S. patent application Ser. No.17/822,251, filed Aug. 25, 2022, which is a continuation in part of PCTPatent Application Serial No. PCT/US2022/073741, filed Jul. 14, 2022,which claims priority to U.S. Provisional Application Ser. No.63/222,916, filed Jul. 16, 2021, both of which are hereby expresslyincorporated by reference in its entirety.

RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

Reference is made to U.S. Provisional Patent Application No. 63/222,916,filed Jul. 16, 2021, U.S. Provisional Patent Application Ser. No.63/053,498 filed Jul. 17, 2020, U.S. Provisional Patent Application Ser.No. 63/222,916, filed Jul. 16, 2021, PCT Patent Application Serial No.PCT/US2021/042079 filed Jul. 16, 2021, U.S. Provisional PatentApplication Ser. No. 63/345,821, filed May 25, 2022, the contents ofwhich are incorporated herein by reference in their entireties.

Reference is made to GB patent application Serial No. 1900858.0, filed22 Jan. 2019, U.S. patent application Ser. No. 62/951,770, filed 20 Dec.2019, International application PCT/GB2020/050120, filed 20 Jan. 2020,and U.S. provisional patent application 63/053,494, filed Jul. 17, 2020.

The foregoing applications, and all documents cited therein or duringtheir prosecution (“appln cited documents”) and all documents cited orreferenced in the appln cited documents, and all documents cited orreferenced herein (“herein cited documents”), and all documents cited orreferenced in herein cited documents, together with any manufacturer'sinstructions, descriptions, product specifications, and product sheetsfor any products mentioned herein or in any document incorporated byreference herein, are hereby incorporated herein by reference, and maybe employed in the practice of the invention. More specifically, allreferenced documents are incorporated by reference to the same extent asif each individual document was specifically and individually indicatedto be incorporated by reference.

REFERENCE TO SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledINSTB.006C3.xml, created on May 3, 2023, which is 266,974 bytes in size.The information in the electronic format of the Sequence Listing isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to a chimeric molecule useful in adoptivecell therapy (ACT), and cells comprising the same. The chimeric moleculecan act as a modulator of cellular activity enhancing responses when anendogenous T-cell receptor (TCR) is engaged with its cognate antigen.The present invention also provides proteins, nucleic acids encoding thechimeric molecule and therapeutic uses thereof.

BACKGROUND OF THE INVENTION

Adoptive cell therapy (ACT) using autologous T-cells to mediate cancerregression has shown much promise in early clinical trials. Severalgeneral approaches have been taken such as the use of naturallyoccurring tumor reactive or tumor infiltrating lymphocytes (TILs)expanded ex vivo. Additionally, T-cells may be genetically modified toretarget them towards defined tumor antigens. This can be done via thegene transfer of peptide (p)-major histocompatibility complex (MHC)specific T-cell Receptors (TCRs) or synthetic fusions between tumorspecific single chain antibody fragment (scFv) and T-cell signalingdomains (e.g. CD3), the latter being termed chimeric antigen receptors(CARs).

TIL and TCR transfer has proven particularly good when targetingmelanoma (Rosenberg et al. 2011; Morgan 2006), whereas CAR therapy hasshown much promise in the treatment of certain B-cell malignancies(Grupp et al. 2013).

Costimulatory signals are useful to achieve robust CAR T cell expansion,function, persistence and antitumor activity. The success of CAR therapyin leukemia has been partly attributed to the incorporation ofcostimulatory domains (e.g. CD28 or CD137) into the CAR construct,signals from which synergize with the signal provided by CD3ζ to enhanceanti-tumor activity. The basis of this observation relates to theclassical signal 1/signal 2 paradigm of T-cell activation. Here signal1, provided by the TCR complex, synergizes with signal 2 provided bycostimulatory receptors such as CD28, CD137 or CD134 to permit the cellsto undergo clonal expansion, IL2 production and long term survivalwithout the activation induced cell death (AICD) associated with signal1 alone. Furthermore the involvement of signal 2 enhances the signalgenerated through signal 1 allowing the cells to respond better to lowavidity interactions such as those encountered during anti-tumorresponses.

Targeted costimulation will have beneficial effects for non-CAR-basedT-cell therapies. For example, incorporating costimulatory domains intoa chimeric TCR has been shown to enhance responses of T-cells towardspMHC (Govers 2014). While tumor infiltrating lymphocytes (TILs) utilizetheir endogenous TCRs to mediate tumor recognition, it has not beenpossible to engineer the endogenous TCR. Thus TIL are subject tosubstantial limitations as tumor cells express very few costimulatoryligands. The ability to induce targeted costimulation of TIL, or indeedany other adoptive T-cell therapy product, would be beneficial.

Citation or identification of any document in this application is not anadmission that such document is available as prior art to the presentinvention.

SUMMARY OF THE INVENTION

Provided herein are chimeric molecules, in particular chimeric proteins,designed to provide costimulation when the endogenous TCR is engagedwith its cognate antigen. Mechanistically, the proposed constructs maybe incorporated in the endogenous TCR complex. When the endogenous TCRcomplex machinery is engaged with their cognate antigen, the TCRreceptor complex aggregates, forcing the clustering of these chimericconstructs. This clustering results in the activation of their signalingdomains, causing an increase in costimulation. This costimulationmanifests itself in a measurable improvement in the effector function ofthe recipient T cell: increased in activation markers, increase cytokinesecretion (IL-2 in particular) and increased proliferation.

Some embodiments herein relate to a chimeric molecule, advantageously achimeric protein, that provides costimulation to the T cell when theendogenous T cell receptor is engaged. This molecule may comprise a TCRclustering domain and a signaling domain that may contain a CD40intracellular domain or signaling fragment thereof.

The TCR clustering domain may be one or more of the proteins typicallyfound in the TCR complex, such as but not limited to, CD3D, CD3E, CD3G,CD3Z, CD3-eta and the constant chains of pre-TCR alpha (PTCRA) TCRalpha, TCR beta, TCR gamma or TCR delta.

The signaling domain may also comprise, an additional full lengthcostimulatory domain, including but not limited to CD2, CD9, CD26, CD27,CD28, CD29, CD38, CD40, CD43, CD46, CD49d, CD55, CD73, CD81, CD82, CD99,CD100, CD134 (OX40), CD137 (41BB), CD150 (SLAM), CD270 (HVEM), CD278(ICOS), CD357 (GITR), or EphB6.

While CD3D, CD3E, CD3G, CD3Z work alone; the constructs containing TCRconstant chains (either alpha/beta or gamma/delta) are preferablyco-expressed with their respective partner in bicistronic configuration:TCR alpha with TCR beta and TCR gamma with TCR delta. Therefore, TCRalpha containing constructs are advantageously co-expressed with TCRbeta and vice versa; and TCR gamma containing constructs should beco-expressed with TCR delta and vice versa. In the context of TILs andany other alpha-beta T cells; the preferred configuration includes TCRgamma-delta; and in gamma-delta T cells, the preferred configurationincludes TCR alpha-beta to minimize interference/disruption with theendogenous TCR machinery and the TCR pairing.

For CD3D, CD3E, CD3G and CD3Z, the transmembrane and extracellularportions are advantageously utilized. However, the present inventionalso contemplates portions or the totality of their intracellularcomponents, which could potentially minimize the disruption of theendogenous TCR complex signaling or help to further amplify theendogenous TCR signaling.

In another aspect, the invention provides a chimeric protein comprisinga clustering domain and a signaling domain that may contain a CD40intracellular domain or signaling fragment thereof. In some embodiments,the clustering domain is capable of oligomerization and/or selfassembly. In some embodiments, clustering comprises formation of ahomodimer or homotrimer. In some embodiments, clustering comprisesoligomerization with a different protein to form a heterodimer orheterotrimer. In some embodiments, the chimeric protein is constitutiveas signaling, for example independent of receptor engagement by anextracellular ligand or independent of receptor engagement by anextracellular ligand attached to a different cell. In some embodiments,the clustering domain comprises a transmembrane domain. In someembodiments, the clustering domain comprises a transmembrane domain andfurther comprises activating mutations that promote dimerization oroligomerization. In some embodiments, the clustering domain comprises anextracellular domain, such as but not limited to an extracellular domainof a receptor. In some embodiments, the clustering domain comprises anextracellular domain of a receptor and further comprises activatingmutations in the extracellular domain that promote dimerization oroligomerization. In some embodiments, the clustering domain comprises aleucine zipper. In some embodiments, the leucine zipper comprises orconstitutes a transmembrane domain. In some embodiments, the leucinezipper comprises or constitutes a soluble domain. Non-limiting examplesof clustering domains include clustering domains of the thrombopoietinreceptor (TpoR), erythropoietin receptor (EpoR), growth hormone receptor(GHR), glycophorin A (GPA) transmembrane domain, and activating mutantsthereof. In some embodiments, clustering may be modulated by a smallmolecule. In some embodiments, clustering may be modulated bypost-translational modifications.

In another aspect, the invention provides a chimeric protein whichcomprises an extracellular ligand binding domain linked to anintracellular signaling domain by a transmembrane domain. In someembodiments, the extracellular ligand binding domain is selected orengineered to bind to an extracellular ligand that maintains two or morecopies of the chimeric protein in proximity to one another such that thesignaling domain is activated. The extracellular ligand binding domainis considered one part of a specific binding pair (sbp) and theextracellular ligand is the second part of the specific binding pair. Insome embodiments, one member of the sbp comprises a protein or receptoror extracellular portion thereof and the second sbp comprises a bindingprotein specific for the first member of the sbp. In some embodiments,the extracellular sbp is bivalent. In some embodiments, theextracellular sbp is trivalent. Nonlimiting examples of extracellularligands include antibodies and bivalent antigen binding fragmentsthereof. Non-limiting examples of extracellular ligand binding domainsof chimeric proteins of the invention (i.e., sbp members) include,without limitation, NKG2A, CD27, CD137, GITR, PD-1, PD-L1, FasL, OX40,CTLA4, ICOS, CD40, EGFR, HER2 and extracellular portions thereof.Complementary sbp members include, without limitation, pembrolizumab forPD1, trastuzumab for HER2, cetuximab for EGFR, tremelimumab for CTLA4,varlilumab for CD27, and urelumab for CD137. In some embodiments, theintracellular signaling domain comprises a CD40 intracellular domain orsignaling fragment thereof.

In some embodiments, the CD40 signaling domain comprises SEQ ID NO:154,SEQ ID NO:155, or SEQ ID NO:156. In some embodiments, the CD40 signalingfragment comprises, consists, or consists essentially of an SH3 motif(KPTNKAPH, PTNKAPHP or PTNKAPH), TRAF2 motif (PKQE, PKQET, PVQE, PVQET,SVQE, SVQET), TRAF6 motif (QEPQEINFP or QEPQEINFP), PKA motif (KKPTNKA,SRISVQE, or a combination thereof, or is a full length CD40intracellular domain. In some embodiments, one or more of the SH3,TRAF2, TRAF6, or PKA motifs of the CD40 signaling domain is mutated. Insome embodiments, one or more of the SH3, TRAF2, TRAF6, or PKA motifs ofthe CD40 signaling domain is present in multiple copies.

Disclosed in this application is an engineered protein. In someembodiments, the engineered protein has at least 80%, 85%, 90%, 95%,96%, 97%, 98%, 99%, 100%, or any integer that is between 80 and 100%,identity to SEQ ID NO: 166, wherein the sequence is not SEQ ID NO: 123.

In some embodiments, the engineered protein has at least 80%, 85%, 90%,95%, 96%, 97%, 98%, 99%, 100%, or any integer that is between 80 and100%, identity to SEQ ID NO: 167, wherein the sequence is not SEQ ID NO:123.

In some embodiments, the engineered protein further comprises a bindingdomain, CD28 domain, and CD40 domain. In some embodiments, theengineered protein further comprises a signal peptide sequence. In someembodiments, the signal peptide sequence has at least 80%, 85%, 90%,95%, 96%, 97%, 98%, 99%, 100%, or any integer that is between 80 and100%, identity to the amino acid sequence of SEQ ID NO: 157. In someembodiments, the binding domain comprises a VL sequence, a VH sequence,and an at least one linker. In some embodiments, the at least one linkerhas at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or anyinteger that is between 80 and 100%, identity to the amino acid sequenceof SEQ ID NO: 159 or 161. In some embodiments, the binding domaincomprises two linker sequences. In some embodiments, the two linkersequences have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, orany integer that is between 80 and 100%, identity to amino acidsequences SEQ ID NO: 159 and SEQ ID NO: 161, respectively. In someembodiments, the VL sequence has at least 80%, 85%, 90%, 95%, 96%, 97%,98%, 99%, 100%, or any integer that is between 80 and 100%, identity tothe amino acid sequence of SEQ ID NO: 158. In some embodiments, the VHsequence has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, orany integer that is between 80 and 100%, identity to the amino acidsequence of SEQ ID NO: 160. In some embodiments, the CD40 domain has atleast 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or any integer thatis between 80 and 100%, identity to the amino acid sequence of SEQ IDNO: 165. In some embodiments, the CD28 domain comprises a CD28transmembrane domain. In some embodiments, the CD28 transmembrane domainhas at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or anyinteger that is between 80 and 100%, identity to SEQ ID NO: 163. In someembodiments, the CD28 domain comprises a CD28 extracellular domain. Insome embodiments, the CD28 extracellular domain has at least 80%, 85%,90%, 95%, 96%, 97%, 98%, 99%, 100%, or any integer that is between 80and 100%, identity to SEQ ID NO: 162. In some embodiments, the CD28domain comprises a CD28 intracellular domain. In some embodiments, theCD28 intracellular domain has at least 80%, 85%, 90%, 95%, 96%, 97%,98%, 99%, 100%, or any integer that is between 80 and 100%, identity toSEQ ID NO: 164. In some embodiments, the protein further comprises 1, 2,3, 4, 5, or all 6 CDR sequence(s) selected from the group consisting of:QASQSLSNLLA (SEQ ID NO: 168), GASNLES (SEQ ID NO: 169), QGGHYSGL (SEQ IDNO: 170), TNDMN (SEQ ID NO: 171), VIYSDDTPDYATWAKG (SEQ ID NO: 172),and/or GHYDSAVYAYALNI (SEQ ID NO: 173). In some embodiments, the bindingdomain and CD28 domain are connected by an at least one linker.

In some embodiments, an engineered protein is provided. It can comprisean amino acid sequence that is at least 80% identical to the amino acidsequence of SEQ ID NO: 166 or 167, wherein the amino acid sequence doesnot include at least one of:

(SEQ ID NO: 174) QKLISEEDLE or (SEQ ID NO: 175)LVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKI.

In some embodiments, a CoStAR is provided. It can comprise: a) anoptional signal peptide; b) a binding domain, wherein the binding domainbinds to an anti-pembrolizumab antibody or binding fragment thereof; c)a CD28 domain; and d) a CD40 domain. Wherein a) is optionally linked tob), wherein b) is linked to c), wherein c) is linked to d), and whereinthe CoStAR comprises an amino acid sequence that: i) lacks at least oneof:

(SEQ ID NO: 174) QKLISEEDLE or (SEQ ID NO: 175)LVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKI;ii) has an amino acid sequence that is greater than 95% identical to SEQID NO: 166 or 167; iii) has an amino acid sequence that is greater than80% identical to SEQ ID NO: 166 or 167 and is not SEQ ID NO: 123; or iv)any combination of i-iv.

In some embodiments, a fusion protein is provided. The fusion proteincomprises a) a means for binding to an antibody that binds topembrolizumab; b) a CD28 domain; and c) CD40 domain. Wherein a) islinked to b), wherein b) is linked to c), and wherein the fusion proteincomprises an amino acid sequence that: i) lacks at least one of:

(SEQ ID NO: 174) QKLISEEDLE or (SEQ ID NO: 175)LVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKI;ii) has an amino acid sequence that is greater than 95% identical to SEQID NO: 166 or 167; iii) has an amino acid sequence that is greater than80% identical to SEQ ID NO: 166 or 167 and is not SEQ ID NO: 123; or iv)any combination of i-iv.

In some embodiments, a fusion protein is provided that comprises theamino acid sequence of SEQ ID NO: 166.

In some embodiments, a fusion protein is provided that comprises theamino acid sequence of SEQ ID NO: 167.

In some embodiments, a nucleic acid which encodes the protein of any oneof the preceding claims.

Also disclosed herein is a nucleic acid which encodes a protein of anyone of the embodiments of the present application.

Also disclosed herein is a vector which comprises a nucleic acid of anyone of the embodiments of the present application.

Also disclosed herein is a cell which expresses a protein of any one ofthe embodiments of the present application.

Also disclosed herein is a cell which expresses at least two proteins ofany one of the embodiments of the present application.

Also disclosed herein is a method of making the cell of any one of theembodiments of the present application which comprises the step oftransducing or transfecting a cell with a vector of any one of theembodiments of the present application.

Also disclosed herein is a method for preparing a population of cellsthat express a protein of any one of the embodiments of the presentapplication, comprising detecting expression of the protein on thesurface of cells transfected or transduced with a vector according toany one of the embodiments of the present application and selectingcells which are identified as expressing the protein.

Also disclosed herein is a cell population produced by the method of anyone of the methods disclosed in the present application.

Also disclosed herein is a cell population which is enriched for cellexpression a protein of any one of the embodiments of the presentapplication.

Also disclosed herein is a method for treating a disease in a subject inneed thereof, which comprises the step of administering the cell of anyone of the embodiments of the present application, or the cellpopulation of any one of the embodiments of the present application, tothe subject.

Accordingly, it is an object of the invention not to encompass withinthe invention any previously known product, process of making theproduct, or method of using the product such that Applicants reserve theright and hereby disclose a disclaimer of any previously known product,process, or method. It is further noted that the invention does notintend to encompass within the scope of the invention any product,process, or making of the product or method of using the product, whichdoes not meet the written description and enablement requirements of theUSPTO (35 U.S.C. § 112, first paragraph) or the EPO (Article 83 of theEPC), such that Applicants reserve the right and hereby disclose adisclaimer of any previously described product, process of making theproduct, or method of using the product. It may be advantageous in thepractice of the invention to be in compliance with Art. 53(c) EPC andRule 28(b) and (c) EPC. All rights to explicitly disclaim anyembodiments that are the subject of any granted patent(s) of applicantin the lineage of this application or in any other lineage or in anyprior filed application of any third party is explicitly reserved.Nothing herein is to be construed as a promise.

It is noted that in this disclosure and particularly in the claimsand/or paragraphs, terms such as “comprises”, “comprised”, “comprising”and the like can have the meaning attributed to it in U.S. patent law;e.g., they can mean “includes”, “included”, “including”, and the like;and that terms such as “consisting essentially of” and “consistsessentially of” have the meaning ascribed to them in U.S. patent law,e.g., they allow for elements not explicitly recited, but excludeelements that are found in the prior art or that affect a basic or novelcharacteristic of the invention.

These and other embodiments are disclosed or are obvious from andencompassed by, the following Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description, given by way of example, but notintended to limit the invention solely to the specific embodimentsdescribed, may best be understood in conjunction with the accompanyingdrawings.

FIG. 1A-1C—Schematic models for universal costimulatory proteins. (FIG.1A) TCR incorporated antigen agnostic receptor (TIAAR) comprisesmodifying components of the TCR complex and associated signalingadaptors. (FIG. 1B) A constitutive costimulatory receptor comprisingtransmembrane domains (TMDs) and features that enable inducible orconstitutive activation. (FIG. 1C) An inducible costimulatory receptorcapable of induction and activation by extracellular ligand binding.

FIG. 2 —Cytokine production by TCR incorporated antigen agnosticreceptor (TIAAR) transduced cells. Cytokine production (Bcl-xL, IL2,IFNg and TNFa) from genetically modified and non-transduced T cells(NTD) of two donors was determined after overnight stimulation witheither Ba/F3 OKT3 targets or left unstimulated (i.e., T cells only).

FIGS. 3A-3B—Proliferation and activation marker expression by TIAARtransduced cells. Proliferation (T cell counts) and activation markerexpression (41BB and CD69) was determined for genetically modified andnon-transduced T cells (NTD) from donor 1 (FIG. 3A) and donor 2 (FIG.3B) after 5-day co-culture with either Ba/F3 OKT3 targets or leftunstimulated (i.e., T cells only).

FIG. 4 —Cytokine production in leucine zipper based universal CoStAR(LZ) transduced cells. Cytokine production (Bcl-xL, IL2, IFNg and TNFa)from genetically modified and non-transduced T cells (NTD) of two donorswas determined after overnight stimulation with either Ba/F3 OKT3targets or left unstimulated (i.e., T cells only).

FIGS. 5A-5B—Proliferation and activation marker expression by LZ-CoStARtransduced cells. Proliferation (T cell counts) and activation markerexpression (41BB and CD69) was determined for genetically modified andnon-transduced T cells (NTD) from donor 1 (FIG. 5A) and donor 2 (FIG.5B) after 5-day co-culture with either Ba/F3 OKT3 targets or leftunstimulated (i.e., T cells only).

FIG. 6 —Cytokine production in inducible universal CoStAR transducedcells. Cytokine production (Bcl-xL, IL2, IFNg and TNFa) from geneticallymodified and non-transduced T cells (NTD) of two donors was determinedafter overnight stimulation with either Ba/F3 OKT3 targets or leftunstimulated (i.e., T cells only). The universal CoStAR is inducible bypembrolizumab.

FIGS. 7A-7B—Proliferation and activation marker expression by inducibleuniversal CoStAR transduced cells. Proliferation (T cell counts) andactivation marker expression (41BB and CD69) was determined forgenetically modified and non-transduced T cells (NTD) from donor 1 (FIG.7A) and donor 2 (FIG. 7B) after 5-day co-culture with either Ba/F3 OKT3targets or left unstimulated (i.e., T cells only). The universal CoStARis inducible by pembrolizumab.

FIG. 8A—is a schematic of a protein of some embodiments provided herein.It is a protein comprising, Universal CoStAR sequence, comprising anoptional section, a binding domain, a CD28 domain, and a CD40 domain.

FIG. 8B—is a schematic of some embodiments provided herein.

FIG. 8C—outlines a set of sequences of some embodiments provided herein.

FIG. 9 —depicts a sequence of an anti-pembrolizumab CoStAR Sequence(“Universal CoStAR”) (SEQ ID NO: 166), containing an optional signaldomain.

FIG. 10 —depicts a sequence of an anti-pembrolizumab CoStAR Sequence(“Universal CoStAR”) (SEQ ID NO: 166), without the optional signaldomain.

FIG. 11 —depicts a sequence alignment between the anti-pembrolizumabCoStAR Sequence (“Universal CoStAR”) (SEQ ID NO: 166) and the vectorclone pIB1102 (SEQ ID NO: 123).

FIG. 12 —depicts the transduction efficiency of constructs into TILsafter 21 days. Round 1 (left panel) and Round 2 (right panel) oftransduction was performed with constructs CTP386.1 and CTP387.1 acrossa variety of TIL organ types (x-axis).

FIG. 13 —depicts the transduction efficiency of constructs into TILsafter 21 days. Round 1 (left panel) and Round 2 (right panel) oftransduction was performed with constructs 322 and 1324 across a varietyof TIL organ types (x-axis).

FIG. 14 —depicts the transduction efficiency of constructs into TILsafter 21 days. Round 1 (left panel) and Round 2 (right panel) oftransduction was performed with construct CTP205 across a variety of TILorgan types (x-axis).

FIGS. 15A-15E—depict the fold-expansion of TILs following a serialstimulation assay. Anti-CEA or anti-FOLR modified TILs from CRC 9823were treated every 7 days with the target K562 OKT3 CEACAM5 or OKT3FOLR, respectively. The readout was measured using the cell count overtime. Shown in panels are the fold-expansion for anti-CEA TILs with K562OKT3 CEACAM5 exposure (FIG. 15A), anti-FOLR TILs with OKT3 FOLR exposure(FIG. 15B), a universal CoStAR with K562 OKT3 CEACAM5 exposure (FIG.15C), a universal CoStAR with K562 OKT3 CEACAM5 exposure and 5 ug/mLpembro (FIG. 15D), and a universal CoStAR with K562 OKT3 CEACAM5exposure and 250 ug/mL pembdro (FIG. 15E).

FIGS. 16A-16E—depict the fold-expansion of TILs following a serialstimulation assay. Anti-CEA or anti-FOLR modified TILs from Mel 11909were treated every 7 days with the target K562 OKT3 CEACAM5 or OKT3FOLR, respectively. The readout was measured using the cell count overtime. Shown in panels are the fold-expansion for anti-CEA TILs with K562OKT3 CEACAM5 exposure (FIG. 16A), anti-FOLR TILs with OKT3 FOLR exposure(FIG. 16B), a universal CoStAR with K562 OKT3 CEACAM5 exposure (FIG.16C), a universal CoStAR with K562 OKT3 CEACAM5 exposure and 5 ug/mLpembro (FIG. 16D), and a universal CoStAR with K562 OKT3 CEACAM5exposure and 250 ug/mL pembdro (FIG. 16E).

FIGS. 17A-17B—depict the increase in IL2 production (pg/mL) in anti-CEA,anti-FOLR, and Universal CoStAR modified TLS from CRC983 (FIG. 17A) orMel 11909 (FIG. 17B).

FIG. 18 —depicts a pie chart of the tumor types (n=15) used as digeststo generate TIL cells in Example 3.

FIG. 19 —depicts an example timeline for transducing CoStAR constructsinto cells using the protocol outlined in Example 3.

FIG. 20 —depicts the transduction efficiency of constructs into TILsafter 1 day. Round 1 (left panel) and Round 2 (right panel) oftransduction was performed with across a variety of TIL organ types(x-axis). Plotted on the y-axis, is the percent of cells with positiveexpression of FOLR1 following transduction.

FIG. 21 —depicts the transduction efficiency of constructs into TILsafter 1 day. Round 1 (left panel) and Round 2 (right panel) oftransduction was performed across a variety of TIL organ types (x-axis).Plotted on the y-axis, is the percent of cells with positive expressionof CEA following transduction.

FIG. 22 —depicts the transduction efficiency of constructs into TILsafter 1 day. Round 1 (left panel) and Round 2 (right panel) oftransduction was performed across a variety of TIL organ types (x-axis).Plotted on the y-axis, is the percent of cells with positive expressionof both FOLR1 and CEA following transduction.

FIG. 23 —depicts the round 1 (left panel) and round 2 (right panel) ofAnti-CEA (386.1) cells that were percent positive for CEA expressionafter pre-sort, post-sort, day 2, and day 4 as outlined in Example 4.

FIG. 24 —depicts the round 1 (left panel) and round 2 (right panel) ofAnti-CEA (387.1) cells that were percent positive for CEA expressionafter pre-sort, post-sort, day 2, and day 4 as outlined in Example 4.

FIG. 25 —depicts the round 1 (left panel) and round 2 (right panel) ofAnti-FOLR cells that were percent positive for FOLR expression afterpre-sort, post-sort, day 2, and day 4 as outlined in Example 4.

FIG. 26 —depicts the round 1 (left panel) and round 2 (right panel) ofUniversal CoStAR (pIB1322) cells that were percent positive for bothFOLR and CEA after pre-sort, post-sort, day 2, and day 4 as outlined inExample 4.

FIG. 27 —depicts the round 1 (left panel) and round 2 (right panel) ofUniversal CoStAR (pIB1324) cells that were percent positive for bothFOLR and CEA after pre-sort, post-sort, day 2, and day 4 as outlined inExample 4.

FIGS. 28A-28D—depict the ratio of CD4 to CD8 positive TIL cells of thoseenriched for FOLR and/or CEA expression after 21 days. FIG. 28A depictsthe ratio in CRC cells, FIG. 28B depicts the ratio in NSCLC cells, FIG.28C depicts the ratio in ovarian cells, and FIG. 28D depicts the ratioin melanoma cells.

FIG. 29 —depicts an example timeline for sorting TIL cells and testingfor function, using the protocol outlined in Example 4.

FIG. 30A-30H—depicts the increase in IFNg production (pg/mL) in TIL andK562 cells lines following co-culturing, across the cell types CRC-11974(FIG. 30A), CRC-11959 (FIG. 30B), NSCLC-9332 (FIG. 30C), NSCLC-9596(FIG. 30D), Ovarian cells (FIG. 30E), Melanoma-CC60 (FIG. 30F),Melanoma-11909 (FIG. 30G), and Melanoma-17614 (FIG. 30H).

FIG. 31A-31H—depicts the increase in IL2 production (pg/mL) in TIL andK562 cells lines following co-culturing, across the cell types CRC-11974(FIG. 31A), CRC-11959 (FIG. 31B), NSCLC-9332 (FIG. 31C), NSCLC-9596(FIG. 31D), Ovarian cells (FIG. 31E), Melanoma-CC60 (FIG. 31F),Melanoma-11909 (FIG. 31G), and Melanoma-17614 (FIG. 31H).

FIG. 32A-32H—depicts the increase in TNFa production (pg/mL) in TIL andK562 cells lines following co-culturing, across the cell types CRC-11974(FIG. 32A), CRC-11959 (FIG. 32B), NSCLC-9332 (FIG. 32C), NSCLC-9596(FIG. 32D), Ovarian cells (FIG. 32E), Melanoma-CC60 (FIG. 32F),Melanoma-11909 (FIG. 32G), and Melanoma-17614 (FIG. 32H).

DETAILED DESCRIPTION OF THE INVENTION

Disclosed herein are a variety of engineered proteins. In someembodiments, the engineered protein that has at least 80%, 85%, 90%,95%, 96%, 97%, 98%, 99%, 100%, or any integer that is between 80 and100%, identity to SEQ ID NO: 166, and wherein the sequence is not SEQ IDNO: 123. In some embodiments, the engineered protein has at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or any integer thatis between 70 and 100%, identity to SEQ ID NO: 166. In some embodiments,the engineered protein has an at least 80% identity to SEQ ID NO:166. Insome embodiments, the engineered protein has an at least 90% identity toSEQ ID NO:166. In some embodiments, the engineered protein is SEQ IDNO:166 (FIG. 9 ). In some embodiments, the sequence is at least 80%identical and is not the sequence of SEQ ID NO: 123.

In some embodiments, the engineered protein that has at least 80%, 85%,90%, 95%, 96%, 97%, 98%, 99%, 100%, or any integer that is between 80and 100%, identity to SEQ ID NO: 167, and wherein the sequence is notSEQ ID NO: 123. In some embodiments, the engineered protein that has atleast 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or anyinteger that is between 70 and 100%, identity to SEQ ID NO: 167. In someembodiments, the engineered protein has an at least 80% identity to SEQID NO:167. In some embodiments, the engineered protein has an at least90% identity to SEQ ID NO:167. In some embodiments, the engineeredprotein is SEQ ID NO:167 (FIG. 10 ). In some embodiments, the sequenceis at least 80% identical, and is not the sequence of SEQ ID NO: 123.

In some embodiments, an engineered protein is provided that has at least80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or any integer that isbetween 80 and 100%, identity to SEQ ID NO: 166, and wherein thesequence is not SEQ ID NO: 123.

In some embodiments, an engineered protein is provided that has at least80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or any integer that isbetween 80 and 100%, identity to SEQ ID NO: 167, and wherein thesequence is not SEQ ID NO: 123.

In some embodiments, the engineered protein, CoStAR or fusion proteinhas a general structure as depicted in FIG. 8A.

In some embodiments, the engineered protein CoStAR or fusion protein hasa general structure as depicted in FIG. 8B.

In some embodiments, the engineered protein CoStAR or fusion proteincomprises at least one sequence depicted in FIG. 8C.

In some embodiments, the arrangement in FIG. 8A is an embodimentseparate from the embodiments in FIGS. 8B and/or 8C. In someembodiments, the arrangement in FIG. 8B is an embodiment separate fromthe embodiments in FIGS. 8C and/or 8A. In some embodiments, thearrangement in FIG. 8C is an embodiment separate from the embodiments inFIGS. 8A and/or 8B (thus, the sequence itself is envisioned, in someembodiments, as the entirety of the engineered protein). In someembodiments, FIG. 8B depicts some embodiments that are a subset of FIG.8C. In some embodiments, FIG. 8C depicts some embodiments that are asubset of FIG. 8A and FIG. 8B. Exemplary CDRs are underlined in FIG. 8C(SEQ ID NO: 158 and 160).

In some embodiments, the engineered protein, CoStAR or fusion protein isas depicted in FIG. 9 or 10 . Exemplary CDRs are underlined in FIG. 9and FIG. 10 . In some embodiments, the engineered protein, CoStAR orfusion protein is different from other fusion proteins, as shown in FIG.11 . In some embodiments, the engineered protein, CoStAR or fusionprotein can lack a tag component and/or a section of CD28.

In some embodiments, the engineered protein comprises a binding domain.In some embodiments, the engineered protein comprises a CD28 domain. Insome embodiments, the engineered protein comprises a CD40 domain. Insome embodiments, the engineered protein comprises 1, 2, or all 3 of abinding domain, a CD28 domain, and/or a CD40 domain.

In some embodiments, the engineered protein comprises a signal peptidesequence. The term “signal peptide” is given its usual scientificmeaning, and thus refers a short peptide that functions in translocatingthe rest of the attached protein. “Signal peptide” thus may also becalled a signal sequence, targeting signal, localization signal,localization sequence, transit peptide, leader sequence or leaderpeptide. It will be understood that the signal peptide may be anypeptide with the function of signaling for the attached peptide to betranslocated to the plasma membrane of a cell. In some embodiments, thesignal peptide sequence has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%,99%, 100%, or any integer that is between 80 and 100%, identity to theamino acid sequence of SEQ ID NO: 157.

In some embodiments, the binding domain comprises 1, 2, or all 3 of a VLsequence, a VH sequence, and/or an at least one linker. In someembodiments, the at least one linker has at least 80%, 85%, 90%, 95%,96%, 97%, 98%, 99%, 100%, or any integer that is between 80 and 100%,identity to the amino acid sequence of SEQ ID NO: 159 or 161. In someembodiments, the binding domain comprises two linker sequences. In someembodiments, the two linker sequences have at least 80%, 85%, 90%, 95%,96%, 97%, 98%, 99%, 100%, or any integer that is between 80 and 100%,identity to amino acid sequences SEQ ID NO: 159 and SEQ ID NO: 161,respectively.

In some embodiments, the VL sequence has at least 80%, 85%, 90%, 95%,96%, 97%, 98%, 99%, 100%, or any integer that is between 80 and 100%,identity to the amino acid sequence of SEQ ID NO: 158. In someembodiments, the VH sequence has at least 80%, 85%, 90%, 95%, 96%, 97%,98%, 99%, 100%, or any integer that is between 80 and 100%, identity tothe amino acid sequence of SEQ ID NO: 160. In some embodiments, all 3 ofthe heavy chain CDRs and/or all three of the light chain CDRs within theVH and VL are identical to the heavy and/or light chain CDRs containedwithin SEQ ID NOs: 158 and 160. In some embodiments, 1, 2, 3, 4, 5 or 6of the CDRs have 1, 2, 3, 4 or more point mutations. In someembodiments, 1, 2, 3, 4, 5, or 6 CDRs are 70, 80, 85, 90, 95, 96, 97,98, 99 or 100% identical to the corresponding CDRs within SEQ ID NOs:158 and/or 160.

In some embodiments, the protein comprises 1, 2, 3, 4, 5, or all 6 CDRsequence(s) selected from the group consisting of: QASQSLSNLLA (SEQ IDNO: 168), GASNLES (SEQ ID NO: 169), QGGHYSGL (SEQ ID NO: 170), TNDMN(SEQ ID NO: 171), VIYSDDTPDYATWAKG (SEQ ID NO: 172), and/orGHYDSAVYAYALNI (SEQ ID NO: 173). In some embodiments, the binding domainand CD28 domain are connected by an at least one linker. In someembodiments, 1, 2, 3, 4, 5 or 6 of the CDRs have 1, 2, 3, 4, or morepoint mutations. In some embodiments, 1, 2, 3, 4, 5, or 6 CDRs are 70,80, 85, 90, 95, 96, 97, 98, 99 or 100% identical to the CDRs.

Also disclosed herein is an engineered protein comprising an amino acidsequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%,or any integer that is between 80 and 100%, identical to the amino acidsequence of SEQ ID NO: 166 or 167.

In some embodiments, an engineered protein is provided that comprises anamino acid sequence that is at least 80% identical to the amino acidsequence of SEQ ID NO: 166 or 167, wherein the amino acid sequence doesnot include at least one of:

(SEQ ID NO: 174) QKLISEEDLE or (SEQ ID NO: 175)LVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKI.

In some embodiments, the CD40 domain has at least 80%, 85%, 90%, 95%,96%, 97%, 98%, 99%, 100%, or any integer that is between 80 and 100%,identity to the amino acid sequence of SEQ ID NO: 165. In someembodiments, the CD28 domain comprises a CD28 transmembrane domain. Insome embodiments, the CD28 transmembrane domain has at least 80%, 85%,90%, 95%, 96%, 97%, 98%, 99%, 100%, or any integer that is between 80and 100%, identity to SEQ ID NO: 163. In some embodiments, the CD28domain comprises a CD28 extracellular domain. In some embodiments, theCD28 extracellular domain has at least 80%, 85%, 90%, 95%, 96%, 97%,98%, 99%, 100%, or any integer that is between 80 and 100%, identity toSEQ ID NO: 162. In some embodiments, the CD28 domain comprises a CD28intracellular domain. In some embodiments, the CD28 intracellular domainhas at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or anyinteger that is between 80 and 100%, identity to SEQ ID NO: 164.

In some embodiments, the amino acid sequence and/or fusion proteinand/or engineered protein does not include at least one of: QKLISEEDLE(SEQ ID NO: 174) orLVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKI (SEQ ID NO: 175). In someembodiments, the engineered protein lacks both of QKLISEEDLE (SEQ ID NO:174) and

(SEQ ID NO: 175) LVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKI.

In some embodiments, the engineered protein comprises a sequence that isat least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or any integerthat is between 80 and 100%, identical to SEQ ID NO: 157.

In some embodiments, the engineered protein comprises a sequence that isat least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or any integerthat is between 80 and 100%, identical to SEQ ID NO: 158.

In some embodiments, the engineered protein comprises a sequence that isat least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or any integerthat is between 80 and 100%, identical to SEQ ID NO: 159.

In some embodiments, the engineered protein comprises a sequence that isat least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or any integerthat is between 80 and 100%, identical to SEQ ID NO: 160.

In some embodiments, the engineered protein comprises a sequence that isat least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or any integerthat is between 80 and 100%, identical to SEQ ID NO: 161.

In some embodiments, the engineered protein comprises a sequence that isat least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or any integerthat is between 80 and 100%, identical to SEQ ID NO: 162.

In some embodiments, the engineered protein comprises a sequence that isat least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or any integerthat is between 80 and 100%, identical to SEQ ID NO: 163.

In some embodiments, the engineered protein comprises a sequence that isat least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or any integerthat is between 80 and 100%, identical to SEQ ID NO: 164.

In some embodiments, the engineered protein comprises a sequence that isat least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or any integerthat is between 80 and 100%, identical to SEQ ID NO: 165.

In some embodiments, the engineered protein comprises 1, 2, 3, 4, 5, 6,7, 8, and/or 9 sequence(s) that have at least 80% identity to SEQ IDNOS: 157-165, respectively. In some embodiments, the engineered proteincomprises 1, 2, 3, 4, 5, 6, 7, 8, and/or 9 sequence(s) that have atleast 90% identity to SEQ ID NOS: 157-165, respectively. In someembodiments, the engineered protein comprises 1, 2, 3, 4, 5, 6, 7, 8,and/or 9 sequence(s) that have at least 95% identity to SEQ ID NOS:157-165, respectively. In some embodiments, the engineered proteincomprises 1, 2, 3, 4, 5, 6, 7, 8, and/or 9 sequence(s) that have atleast 98% identity to SEQ ID NOS: 157-165, respectively. In someembodiments, the engineered protein comprises 1, 2, 3, 4, 5, 6, 7, 8,and/or 9 sequence(s) selected from the group consisting of: SEQ ID NOS:157-165.

Also disclosed herein is a CoStAR. In some embodiments, the CoStARcomprises an optional signal peptide, a binding domain, wherein thebinding domain binds to an anti-pembrolizumab antibody or bindingfragment thereof, a CD28 domain, and a CD40 domain, wherein the signalpeptide is optionally linked to the binding domain, wherein the bindingdomain is linked to the CD28 domain, wherein the CD28 domain is linkedto the CD40 domain, and wherein the CoStAR comprises an amino acidsequence that: i) lacks at least one of: QKLISEEDLE (SEQ ID NO: 174) orLVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKI (SEQ ID NO: 175); ii) has anamino acid sequence that is greater than 95% identical to SEQ ID NO: 166or 167; iii) has an amino acid sequence that is greater than 80%identical to SEQ ID NO: 166 or 167 and is not SEQ ID NO: 123; or iv) anycombination of i-iv.

Also disclosed herein is a fusion protein. In some embodiments, thefusion protein comprises a means for binding to an antibody that bindsto pembrolizumab, a CD28 domain, and a CD40 domain, wherein the meansfor binding to an antibody is linked to a CD28 domain, wherein the CD28domain is linked to the CD40 domain, and wherein the fusion proteincomprises an amino acid sequence that: i) lacks at least one of:QKLISEEDLE (SEQ ID NO: 174) orLVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKI (SEQ ID NO: 175); ii) has an aminoacid sequence that is greater than 95% identical to SEQ ID NO: 166 or167; iii) has an amino acid sequence that is greater than 80% identicalto SEQ ID NO: 166 or 167 and is not SEQ ID NO: 123; or iv) anycombination of i-iv. In some embodiments, the means for binding topembrolizumab is an anti-pembrolizumab antibody. In some embodiments,the anti-pembrolizumab antibody is

In some embodiments, the binding domain or the means for binding to anantibody that binds to pembrolizumab comprises: 1, 2, 3, 4, 5, or all 6CDR sequence(s) selected from the group consisting of: QASQSLSNLLA (SEQID NO: 168), GASNLES (SEQ ID NO: 169), QGGHYSGL (SEQ ID NO: 170), TNDMN(SEQ ID NO: 171), VIYSDDTPDYATWAKG (SEQ ID NO: 172), and/orGHYDSAVYAYALNI (SEQ ID NO: 173). In some embodiments, 1, 2, 3, 4, 5 or 6of the CDRs have 1, 2, 3, 4, or more point mutations. In someembodiments, 1, 2, 3, 4, 5, or 6 CDRs are 70, 80, 85, 90, 95, 96, 97,98, 99 or 100% identical to the CDRs.

In some embodiments, the CD28 domain comprises: SEQ ID Nos: 162, 163,and 164, or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%,98%, 99%, 100%, or any integer that is between 80 and 100%, identicalthereto. In some embodiments, the CD40 domain comprises: SEQ ID No: 165,or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%,100%, or any integer that is between 80 and 100%, identical thereto.

Also disclosed herein is a fusion protein comprising the amino acidsequence of SEQ ID NO: 166. In some embodiments, the fusion proteincomprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%,99%, 100%, or any integer that is between 80 and 100%, identical to SEQID NO: 166. Also disclosed herein is a fusion protein comprising theamino acid sequence of SEQ ID NO: 167. In some embodiments, the fusionprotein comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%,97%, 98%, 99%, 100%, or any integer that is between 80 and 100%,identical to SEQ ID NO: 167.

Also disclosed herein is a nucleic acid which encodes a protein of anyone of the embodiments of the present application.

Also disclosed herein is a vector which comprises a nucleic acid of anyone of the embodiments of the present application.

Also disclosed herein is a cell which expresses a protein of any one ofthe embodiments of the present application.

Also disclosed herein is a cell which expresses at least two proteins ofany one of the embodiments of the present application.

Also disclosed herein is a method of making the cell of any one of theembodiments of the present application which comprises the step oftransducing or transfecting a cell with a vector of any one of theembodiments of the present application.

Also disclosed herein is a method for preparing a population of cellsthat express a protein of any one of the embodiments of the presentapplication, comprising detecting expression of the protein on thesurface of cells transfected or transduced with a vector according toany one of the embodiments of the present application and selectingcells which are identified as expressing the protein.

Also disclosed herein is a cell population produced by the method of anyone of the methods disclosed in the present application.

Also disclosed herein is a cell population which is enriched for cellexpression a protein of any one of the embodiments of the presentapplication.

Also disclosed herein is a method for treating a disease in a subject inneed thereof, which comprises the step of administering the cell of anyone of the embodiments of the present application, or the cellpopulation of any one of the embodiments of the present application, tothe subject.

As used herein, “full length protein” or “full length receptor” refersto a receptor protein, such as, for example, a CD40 receptor protein.The term “full length” encompasses receptor proteins lacking up to about5 or up to 10 amino acids, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10amino acids, at the N-terminal of the mature receptor protein once itssignal peptide has been cleaved. For instance, while a specific cleavagesite of a receptors N-terminal signal peptide may be defined,variability in exact point of cleavage has been observed. The term “fulllength” does not imply presence or absence of amino acids of thereceptors N-terminal signal peptide. In one embodiment, the term “fulllength” (e.g. a full length CD28 or a full length CD40 intracellulardomain, according to certain aspects of the invention) encompassesmature receptor proteins (e.g. CD28 according to certain aspects of theinvention) lacking the N terminal signal peptide lacking up to about 5,for example 1, 2, 3, 4, 5, or up to 10 amino acids at the N-terminal ofthe mature receptor protein once its signal peptide has been cleaved. Asmentioned above, a “full length” CD28 receptor or other receptor or TCRclustering domain according to the various aspects of the invention doesnot include the signal peptide and may lack up to about 5, for example1, 2, 3, 4, 5, or up to 10 amino acids at the N-terminal of the maturereceptor protein (e.g. N terminal residues N, K, I, L and/or V). This isshown in the exemplary fusions, e.g. SEQ ID Nos. 4-12 (note that thesemay lack up to about 5, for example 1, 2, 3, 4, 5, or up to 10 aminoacids at the N-terminal of the mature receptor protein as shown in theboxed region).

The chimeric protein of the present invention may comprise a TCRclustering domain as well as a signaling domain that advantageously maycomprise a CD40 intracellular domain.

The term “T cell receptor,” or “TCR,” refers to a heterodimeric receptorcomposed of αβ or γδ chains that pair on the surface of a T cell. Eachα, β, γ, and δ chain is composed of two Ig-like domains: a variabledomain (V) that confers antigen recognition through the complementaritydetermining regions (CDR), followed by a constant domain I that isanchored to cell membrane by a connecting peptide and a Transmembral TM)region. The TM region associates with the invariant subunits of the CD3signaling apparatus. Each of the V domains has three CDRs. These CDRsinteract with a complex between an antigenic peptide bound to a proteinencoded by the major histocompatibility complex (pMHC) (Davis andBjorkman (1988) Nature, 334, 395-402; Davis et al. (1998) Annu RevImmunol, 16, 523-544; Murphy (2012), xix, 868 p.).

Costimulatory receptor proteins useful in the chimeric proteins of theinvention include, without limitation, CD2, CD9, CD26, CD27, CD28, CD29,CD38, CD40, CD43, CD46, CD49d, CD55, CD73, CD81, CD82, CD99, CD100,CD134 (OX40), CD137 (41BB), CD150 (SLAM), CD270 (HVEM), CD278 (ICOS),CD357 (GITR), or EphB6, which in their natural form compriseextracellular ligand binding domains and intracellular signaltransducing domains. For example, CD2 is characterized as a celladhesion molecule found on the surface of T cells and is capable ofinitiating intracellular signals necessary for T cell activation. CD27is characterized as a type II transmembrane glycoprotein belonging tothe TNFR superfamily (TNFRSF) whose expression on B cells is induced byantigen-receptor activation in B cells. CD28 is one of the proteins on Tcells and is the receptor for CD80 (B7.1) and CD86 (B7.2) ligands onantigen-presenting cells. CD137 (4-1BB) ligand is found on mostleukocytes and on some non-immune cells. OX40 ligand is expressed onmany antigen-presenting cells such as DC2s (dendritic cells),macrophages, and B lymphocytes. In one embodiment, the costimulatoryreceptor protein is full length CD28 as defined herein.

CD40 is a member of the tumor necrosis factor receptor (TNFR)superfamily and several isoforms are generated by alternative splicing.Its ligand, CD154 (also called CD40L) is a protein that is primarilyexpressed on activated T cells. For reference, the human CD40 isoform 1protein sequence is set forth in GenBank accession No. NP_001241.1,including signal peptide (amino acids 1-20), transmembrane domain (aminoacids 194-215), and cytoplasmic domain (amino acids 216-277) (SEQ IDNO:22). CD40 receptor signaling involves adaptor proteins including butnot limited to TNF receptor-associated factors (TRAF), and thecytoplasmic domain comprises signaling components, including but notlimited to an SH3 motif (KPTNKAPH), TRAF2 motif (PKQE, PVQE, SVQE),TRAF6 motif (QEPQEINFP) and PKA motif (KKPTNKA, SRISVQE). Further motifsfor binding to TRAF1, TRAF2, TRAF3, and TRAF5 comprise the majorconsensus sequence (P/S/A/T)X(Q/E)E or minor consensus sequence PXQXXDand can be identified in or obtained from, without limitation, TNFRfamily members such as CD30, Ox40, 4-1BB, and the EBV oncoprotein LMP1.(See, e.g., Ye, H et al., The Structural Basis for the Recognition ofDiverse Receptor Sequences by TRAF2. Molecular Cell, 1999; 4(3):321-30.doi: 10.1016/S1097-2765(00)80334-2; Park H H, Structure of TRAF Family:Current Understanding of Receptor Recognition. Front. Immunol. 2018;9:1999. doi: 10.3389/fimmu.2018.01999).

Examples disclosed herein demonstrate operation of CD40 as a signalingdomain and further that cytokine and chemokine expression profiles arealtered by signaling domain selection. In this regard, the CD40signaling domains of the invention provide distinct and overlappingresponses induced by the different factor binding sites. (See, e.g.,Ahonen, C L et al., The CD40-TRAF6 axis controls affinity maturation andthe generation of long-lived plasma cells. Nat Immunol. 2002; 3:451-456; Mackey M F et al., Distinct contributions of different CD40TRAF binding sites to CD154-induced dendritic cell maturation and IL-12secretion. Eur J Immunol. 2003; 33: 779-789; Mukundan L et al., TNFreceptor-associated factor 6 is an essential mediator of CD40-activatedproinflammatory pathways in monocytes and macrophages. J Immunol. 2005;174: 1081-1090.

In some embodiments, a chimeric protein of the invention comprisessubstantially all of a CD40 costimulatory domain. In some embodiments, achimeric protein of the invention comprises two or more CD40costimulatory domains. In some embodiments, a chimeric protein of theinvention comprises a CD40 costimulatory domain signaling component ormotif, including but not limited to an SH3 motif (KPTNKAPH), TRAF2 motif(PKQE, PVQE, SVQE), TRAF3 motif, TRAF6 motif (QEPQEINFP) or PKA motif(KKPTNKA, SRISVQE) as well as two or more, or three or more, or four ormore such components of motifs, which can be in multiple copies andarranged in any order. In some embodiments, a chimeric protein of theinvention comprises a CD40 costimulatory domain and a CD40 costimulatorydomain signaling component or motif.

In some embodiments, selection of one or more costimulatory domainsignaling component or motif is guided by the cell in which the chimericprotein is to be expressed and/or a desired costimulatory activity moreclosely identified with a signaling component or motif, or avoidance ofa costimulatory activity more closely identified with a signalingcomponent or motif.

In some embodiments, a chimeric protein signaling domain comprises, inaddition to a CD40 costimulatory domain or signaling component or motifthereof, or two or more such domains or components or motifs orcombinations thereof, an additional full length costimulatory domain orsignaling component thereof from, without limitation, CD2, CD9, CD26,CD27, CD28, CD29, CD38, CD40, CD43, CD46, CD49d, CD55, CD73, CD81, CD82,CD99, CD100, CD134 (OX40), CD137 (41BB), CD150 (SLAM), CD270 (HVEM),CD278 (ICOS), CD357 (GITR), or EphB6,

For reference, the human CD28 protein sequence is set forth in GenBankaccession No. NP_006130.1, including signal peptide (amino acids 1-18),extracellular domain (amino acids 19-152), transmembrane domain (aminoacids 153-179) and cytoplasmic domain (amino acids 180-200). Theextracellular domain includes an immunoglobulin type domain (amino acids21-136) which contains amino acids with compose the antigen binding siteand amino acids that form the homodimer interface. The extracellulardomain includes several asparagine residues which may be glycosylated,and the intracellular domain comprises serine and tyrosine residues,which may be phosphorylated.

For reference, the human CD8 alpha chain protein sequence is set forthby GenBank accession No. NP_001139345.1, including signal peptide (aminoacids 1-21), extracellular domain (amino acids 22-182), transmembranedomain (amino acids 183-203), and cytoplasmic domain (amino acids204-235). The extracellular domain includes an immunoglobulin typedomain (amino acids 28-128) which contains amino acids with compose theantigen binding site and amino acids that form the homodimer interface.The extracellular domain includes several asparagine residues which maybe glycosylated, and the intracellular domain comprises serine andtyrosine residues, which may be phosphorylated.

For reference, the human IgG4 constant region sequence is set forth inUniProtKB/Swiss-Prot: accession No. P01861.1, including CH1 (amino acids1-98), hinge (amino acids 99-110), CH2 (amino acids 111-220), CH3 (aminoacids 221-327). The CH2 region includes asparagine at amino acid 177,which is the glycosylated and associated with Fc receptor andantibody-dependent cell-mediated cytotoxicity (ADCC).

For reference, the protein sequence of human CD137 (41BB), another TNFRsuperfamily member, is set forth by GenBank accession No. NP_001552.2,including signal peptide (amino acids 1-23), extracellular domain (aminoacids 24-186), transmembrane domain (amino acids 187-213), andcytoplasmic domain (amino acids 214-255).

For reference, the human CD134 (OX40) protein sequence is set forth byGenBank accession No. NP_003318.1, including signal peptide (amino acids1-28), extracellular domain (amino acids 29-214), transmembrane domain(amino acids 215-235), and cytoplasmic domain (amino acids 236-277).This receptor has been shown to activate NF-kappaB through itsinteraction with adaptor proteins TRAF2 and TRAF5 and studies suggestthat this receptor promotes expression of apoptosis inhibitors BCL2 andBCL21L1/BCL2-XL.

The human T-cell surface antigen CD2 has at least two isoforms. Forreference, the human CD2 isoform1 protein sequence is set forth byNP_001315538.1, including signal peptide (amino acids 1-24),extracellular domain (amino acids 25-235), transmembrane domain (aminoacids 236-261), and cytoplasmic domain (amino acids 262-377). The humanCD2 isoform2 protein sequence is set forth by NP_001758.2

For reference, the human CD357 (GITR) isoform-1 protein sequence is setforth by GenBank accession No. NP_004186.1, including signal peptide(amino acids 1-25), extracellular domain (amino acids 26-162),transmembrane domain (amino acids 163-183), and cytoplasmic domain(amino acids 184-241).

For reference, the human CD29 (beta1 integrin) protein sequence is setforth by GenBank accession No. NP_596867, including signal peptide(amino acids 1-20), extracellular domain (amino acids 21-728),transmembrane domain (amino acids 729-751), and cytoplasmic domain(amino acids 752-798).

The human CD150 (SLAM) protein sequence has at several isoforms. Inaddition to the transmembrane form of CD150 (mCD150), cells ofhematopoietic lineage express mRNA encoding the secreted form of CD150(sCD150), which lacks the entire transmembrane region of 30 amino acids.For reference, human SLAM isoform b is set forth by GenBank accessionNo. NP_003028.1, including signal peptide (amino acids 1-20),extracellular domain (amino acids 21-237), transmembrane domain (aminoacids 238-258), and cytoplasmic domain (amino acids 259-335). Human SLAMisoform a is set forth by GenBank accession No. NP_001317683.1.

In embodiments of the invention, a chimeric protein may be expressedalone under the control of a promoter in a therapeutic population ofcells that have therapeutic activity, for example, Tumor InfiltratingLymphocytes (TILs). Alternatively, the chimeric protein may be expressedalong with a therapeutic transgene such as a chimeric antigen receptor(CAR) and/or T-cell Receptor (TCR). Suitable TCRs and CARs are wellknown in the literature, for example HLA-A*02-NYESO-1 specific TCRs(Rapoport et al. Nat Med 2015) or anti-CD19scFv.CD3ζ fusion CARs(Kochenderfer et al. J Clin Oncol 2015) which have been successfullyused to treat Myeloma or B-cell malignancies respectively. The chimericproteins described herein may be expressed with any known CAR or TCRthus providing the cell with a regulatable growth switch to allow cellexpansion in-vitro or in-vivo, and a conventional activation mechanismin the form of the TCR or CAR for anti-cancer activity. Thus theinvention provides a cell for use in adoptive cell therapy comprising achimeric protein as described herein and a TCR and/or CAR thatspecifically binds to a tumor associated antigen. An exemplary chimericprotein comprising CD28 includes an extracellular antigen binding domainand an extracellular, transmembrane and intracellular signaling domain.

A chimeric protein of the invention optionally comprises a spacer regionbetween the TCR clustering domain and the costimulatory receptor. Asused herein, the term “spacer” refers to the extracellular structuralregion of a chimeric protein that separates the TCR clustering domainfrom the signaling domain of the chimeric protein. In some embodimentslong spacers are employed, for example to target membrane-proximalepitopes or glycosylated antigens (see Guest R. D. et al. The role ofextracellular spacer regions in the optimal design of chimeric immunereceptors: evaluation of four different scFvs and antigens. J.Immunother. 2005; 28:203-211; Wilkie S. et al., Retargeting of human Tcells to tumor-associated MUC1: the evolution of a chimeric antigenreceptor. J. Immunol. 2008; 180:4901-4909). In other embodiments,chimeric proteins bear short spacers, for example to target membranedistal epitopes (see Hudecek M. et al., Receptor affinity andextracellular domain modifications affect tumor recognition byROR1-specific chimeric antigen receptor T cells. Clin. Cancer Res. 2013;19:3153-3164; Hudecek M. et al 27acarbazine27nalling extracellularspacer domain of chimeric antigen receptors is decisive for in vivoantitumor activity. Cancer Immunol. Res. 2015; 3:125-135). In someembodiments, the spacer comprises all or part of or is derived from anIgG hinge, including but not limited to IgG1, IgG2, or IgG4. By “derivedfrom an Ig hinge” is meant a spacer comprising insertions, deletions, ormutations in an IgG hinge. In some embodiments, a spacer can compriseall or part of one or more antibody constant domains, such as but notlimited to CH2 and/or CH3 domains. In some embodiments, in a spacercomprising all or part of a CH2 domain, the CH2 domain is modified so asnot to bind to an Fc receptor. For example, Fc receptor binding inmyeloid cells has been found to impair CAR T cell functionality. In someembodiments, the spacer comprises all or part of an Ig-like hinge fromCD28, CD8, or other protein comprising a hinge region. In someembodiments of the invention that comprise a spacer, the spacer is from1 and 50 amino acids in length.

In some embodiments, the chimeric protein extracellular domain comprisesa linker. Linkers comprise short runs of amino acids used to connectdomains, for example a binding domain with a spacer or transmembranedomain. In order for there to be flexibility to bind ligand, a ligandbinding domain will usually be connected to a spacer or a transmembranedomain by flexible linker comprising from about 5 to 25 amino acids,such as, for example, AAAGSGGSG or GGGGSGGGGSGGGGS. In some embodiments,a chimeric protein comprises a TCR clustering domain joined directly toa signaling domain by a linker, and without a spacer. In someembodiments, a chimeric protein comprises a binding domain joineddirectly to a transmembrane by a spacer and without a linker.

As discussed above, in some embodiments, a chimeric protein comprises afull length primary costimulatory receptor which can comprise anextracellular ligand binding and intracellular signaling portion of,without limitation, CD2, CD9, CD26, CD27, CD28, CD29, CD38, CD40, CD43,CD46, CD49d, CD55, CD73, CD81, CD82, CD99, CD100, CD134 (OX40), CD137(41BB), CD150 (SLAM), CD270 (HVEM), CD278 (ICOS), CD357 (GITR), orEphB6. In other embodiments, the chimeric protein, for instance maycomprise an extracellular ligand binding domain of one of theaforementioned proteins and an intracellular signaling domain of anotherof the aforementioned proteins. In some embodiments, the signalingportion of the chimeric protein comprises a single signaling domain. Inother embodiments, the signaling portion of the chimeric proteincomprises a second intracellular signaling domain such as but notlimited to: CD2, CD27, CD28, CD40, CD134 (OX40), CD137 (4-1BB), CD150(SLAM). In some embodiments, the first and second intracellularsignaling domains are the same. In other embodiments, the first andsecond intracellular signaling domains are different. In someembodiments, the costimulatory receptor is capable of dimerization.Without being bound by theory, it is thought that chimeric proteinsdimerize or associate with other accessory molecules for signalinitiation. In some embodiments, chimeric proteins dimerize or associatewith accessory molecules through transmembrane domain interactions. Insome embodiments, dimerization or association with accessory moleculesis assisted by costimulatory receptor interactions in the intracellularportion, and/or the extracellular portion of the costimulatory receptor.

Although the main function of the transmembrane is to anchor thechimeric protein in the T cell membrane, in some embodiments, thetransmembrane domain influences chimeric protein function. In someembodiments, the transmembrane domain is comprised by the full lengthprimary costimulatory receptor domain. In embodiments of the inventionwherein the chimeric protein construct comprises an extracellular domainof one receptor and an intracellular signaling domain of a secondreceptor, the transmembrane domain can be that of the extracellulardomain or the intracellular domain. In some embodiments, thetransmembrane domain is from CD4, CD8a, CD28, or ICOS. Gueden et al.associated use of the ICOS transmembrane domain with increased CART cellpersistence and overall anti-tumor efficacy (Guedan S. et al., EnhancingCART cell persistence through ICOS and 4-1BB costimulation. JCI Insight.2018; 3:96976). In an embodiment, the transmembrane domain comprises ahydrophobic α helix that spans the cell membrane.

In some embodiments, amino acid sequence variants of the TCR clusteringdomain or other moieties provided herein are contemplated. For example,it may be desirable to improve the binding affinity and/or otherbiological properties of the moiety. Amino acid sequence variants of anantibody moiety may be prepared by introducing appropriate modificationsinto the nucleotide sequence encoding the clustering moiety, or bypeptide synthesis. Such modifications include, for example, deletionsfrom, and/or insertions into and/or substitutions of residues within theamino acid sequences of the antibody moiety. Any combination ofdeletion, insertion, and substitution can be made to arrive at the finalconstruct, provided that the final construct possesses the desiredcharacteristics.

In some embodiments, TCR clustering domain moieties comprising one ormore amino acid substitutions, deletions, or insertions are provided.Amino acid substitutions may be introduced into a binding domain ofinterest and the products screened for a desired activity, e.g.,retained/improved clustering or decreased immunogenicity. In someembodiments, amino acid substitutions may be introduced into one or moreof the primary co-stimulatory receptor domain (extracellular orintracellular), secondary costimulatory receptor domain, orextracellular co-receptor domain. Accordingly, the invention encompasseschimeric proteins and component parts particularly disclosed herein aswell as variants thereof, i.e. chimeric proteins and component partshaving at least 75%, at least 80%, at least 85%, at least 87%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99% sequenceidentity to the amino acid sequences particularly disclosed herein. Theterms “percent similarity,” “percent identity,” and “percent homology”when referring to a particular sequence are used as set forth in theUniversity of Wisconsin GCG software program BestFit. Other algorithmsmay be used, e.g. BLAST, psiBLAST or TBLASTN (which use the method ofAltschul et al. (1990) J. Mol. Biol. 215: 405-410), FASTA (which usesthe method of Pearson and Lipman (1988) PNAS USA 85: 2444-2448).

Particular amino acid sequence variants may differ from a referencesequence by insertion, addition, substitution or deletion of 1 aminoacid, 2, 3, 4, 5-10, 10-20 or 20-30 amino acids. In some embodiments, avariant sequence may comprise the reference sequence with 1, 2, 3, 4, 5,6, 7, 8, 9, 10 or more residues inserted, deleted or substituted. Forexample, 5, 10, 15, up to 20, up to 30 or up to 40 residues may beinserted, deleted or substituted.

In some preferred embodiments, a variant may differ from a referencesequence by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservativesubstitutions. Conservative substitutions involve the replacement of anamino acid with a different amino acid having similar properties. Forexample, an aliphatic residue may be replaced by another aliphaticresidue, a non-polar residue may be replaced by another non-polarresidue, an acidic residue may be replaced by another acidic residue, abasic residue may be replaced by another basic residue, a polar residuemay be replaced by another polar residue or an aromatic residue may bereplaced by another aromatic residue. Conservative substitutions may,for example, be between amino acids within the following groups:

Conservative substitutions are shown in Table 1 below.

TABLE 1 Original Exemplary Preferred Residue Substitutions SubstitutionsIArg (R) Lys; Gln; Asn Lys Asn (N) Gln; His; Asp; Lys; Arg Gln Asp (D)Glu; AsnIu Cys (C) Ser; Ala Ser Gln (Q) Asn; I Asn Glu (E) Asp; Gln AspGly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met;Ala; Phe; Norleucine Leu Leu (L) Norleucinne; Ile; Val; Met; Ala; PheIle Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu Phe (F) Trp;Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Val;Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) Ile; Le u; Met; Phe; Ala; Norleucine Leu

Amino acids may be grouped into different classes according to commonside-chain properties: a. hydrophobic: Norleucine, Met, Ala, Val, Leu,Ile; b. neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; c. acidic: Asp,Glu; d. basic: His, Lys, Arg; e. residues that influence chainorientation: Gly, Pro; aromatic: Trp, Tyr, Phe. Non-conservativesubstitutions will entail exchanging a member of one of these classesfor another class.

The cells used in the present invention may be any lymphocyte that isuseful in adoptive cell therapy, such as a T-cell or a natural killer(NK) cell, an NKT cell, a gamma/delta T-cell or T regulatory cell. Thecells may be allogeneic or autologous to the patient.

T cells or T lymphocytes are a type of lymphocyte that have a centralrole in cell-mediated immunity. They can be distinguished from otherlymphocytes, such as B cells and natural killer cells (NK cells), by thepresence of a T-cell receptor (TCR) on the cell surface. There arevarious types of T cell, as summarized below. Cytotoxic T cells (TCcells, or CTLs) destroy virally infected cells and tumor cells, and arealso implicated in transplant rejection. CTLs express the CD8 moleculeat their surface.

These cells recognize their targets by binding to antigen associatedwith MHC class I, which is present on the surface of all nucleatedcells. Through IL-10, adenosine and other molecules secreted byregulatory T cells, the CD8+ cells can be inactivated to an anergicstate, which prevent autoimmune diseases such as experimental autoimmuneencephalomyelitis.

Memory T cells are a subset of antigen-specific T cells that persistlong-term after an infection has resolved. They quickly expand to largenumbers of effector T cells upon re-exposure to their cognate antigen,thus providing the imm“ne sys”em with “memory” against past infections.Memory T cells comprise three subtypes: central memory T cells (TCMcells) and two types of effector memory T cells (TEM cells and TEMRAcells). Memory cells may be either CD4+ or CD8+. Memory T cellstypically express the cell surface protein CD45RO. Regulatory T cells(Treg cells), formerly known as suppressor T cells, are crucial for themaintenance of immunological tolerance. Their major role is to shut downT cell-mediated immunity toward the end of an immune reaction and tosuppress auto-reactive T cells that escaped the process of negativeselection in the thymus.

Two major classes of CD4⁺ Treg cells have been described—naturallyoccurring Treg cells and adaptive Treg cells. Naturally occurring Tregcells (also known as CD4⁺CD25⁺FoxP3⁺ Treg cells) arise in the thymus andhave been linked to interactions between developing T cells with bothmyeloid (CD11c⁺) and plasmacytoid (CD123⁺) dendritic cells that havebeen activated with TSLP. Naturally occurring Treg cells can bedistinguished from other T cells by the presence of an intracellularmolecule called FoxP3. Adaptive Treg cells (also known as Tr1 cells orTh3 cells) may originate during a normal immune response.

Natural Killer Cells (or NK cells) are a type of cytolytic cell whichform part of the innate immune system. NK cells provide rapid responsesto innate signals from virally infected cells in an MHC independentmanner. NK cells (belonging to the group of innate lymphoid cells) aredefined as large granular lymphocytes (LGL) and constitute the thirdkind of cells differentiated from the common lymphoid progenitorgenerating B and T lymphocytes.

In some embodiments, therapeutic cells of the invention compriseautologous cells engineered to express a chimeric protein. In someembodiments, therapeutic cells of the invention comprise allogeneiccells engineered to express a chimeric protein. Autologous cellsexpressing chimeric proteins may be advantageous in avoidinggraft-versus-host disease (GVHD) due to TCR-mediated recognition ofrecipient alloantigens.

An aspect of the invention provides a nucleic acid sequence of theinvention, encoding any of the chimeric proteins, polypeptides, orproteins described herein (including functional portions and functionalvariants thereof). As used herein, the terms “polynucleotide”,“nucleotide”, and “nucleic acid” are intended to be synonymous with eachother. It will be understood by a skilled person that numerous differentpolynucleotides and nucleic acids can encode the same polypeptide as aresult of the degeneracy of the genetic code. In addition, it is to beunderstood that skilled persons may, using routine techniques, makenucleotide substitutions that do not affect the polypeptide sequenceencoded by the polynucleotides described here to reflect the codon usageof any particular host organism in which the polypeptides are to beexpressed, e.g. codon optimization. Nucleic acids according to theinvention may comprise DNA or RNA. They may be single stranded ordouble-stranded. They may also be polynucleotides which include withinthem synthetic or modified nucleotides. A number of different types ofmodification to oligonucleotides are known in the art. These includemethylphosphonate and phosphorothioate backbones, addition of acridineor polylysine ‘chains at’ the 3′ and/or 5′ ends of the molecule. For thepurposes of the present invention, it is to be understood that thepolynucleotides may be modified by any method available in the art. Suchmodifications may be carried out in order to enhance the in vivoactivity or life span of polynucleotides of interest.

The terms “variant”, “homologue” or “derivative” in relation to anucleotide sequence include any substitution of, variation of,modification of, replacement of, deletion of or addition of one (ormore) nucleic acid from or to the sequence.

The nucleic acid sequence may encode the protein sequence shown as SEQID NO:2 or a variant thereof. The nucleotide sequence may comprise acodon optimized nucleic acid sequence shown engineered for expression inhuman cells.

The invention also provides a nucleic acid sequence which comprises anucleic acid sequence encoding a chimeric protein and a further nucleicacid sequence encoding a T-cell receptor (TCR) and/or chimeric antigenreceptor (CAR).

The nucleic acid sequences may be joined by a sequence allowingco-expression of the two or more nucleic acid sequences. For example,the construct may comprise an internal promoter, an internal ribosomeentry sequence (IRES) sequence or a sequence encoding a cleavage site.The cleavage site may be self-cleaving, such that when the polypeptideis produced, it is immediately cleaved into the discrete proteinswithout the need for any external cleavage activity. Variousself-cleaving sites are known, including the Foot- and Mouth diseasevirus (FMDV) and the 2A self-cleaving peptide. The co-expressingsequence may be an internal ribosome entry sequence (IRES). Theco-expressing sequence may be an internal promoter.

In an aspect, the present invention provides a vector which comprises anucleic acid sequence or nucleic acid construct of the invention.

Such a vector may be used to introduce the nucleic acid sequence(s) ornucleic acid construct(s) into a host cell so that it expresses one ormore chimeric protein(s) according to the first aspect of the inventionand, optionally, one or more other proteins of interest (POI), forexample a TCR or a CAR. The vector may, for example, be a plasmid or aviral vector, such as a retroviral vector or a lentiviral vector, or atransposon-based vector or synthetic mRNA.

The nucleic acids of the present invention may also be used for nucleicacid immunization and gene therapy, using standard gene deliveryprotocols. Methods for gene delivery are known in the art. See, e.g.,U.S. Pat. Nos. 5,399,346, 5,580,859, 5,589,466, incorporated byreference herein in their entireties.

Vectors derived from retroviruses, such as the lentivirus, are suitabletools to achieve long-term gene transfer since they allow long-term,stable integration of a transgene or transgenes and its propagation indaughter cells. The vector may be capable of transfecting or transducinga lymphocyte including a T cell or an NK cell. The present inventionalso provides vectors in which a nucleic acid of the present inventionis inserted. The expression of natural or synthetic nucleic acidsencoding a chimeric protein, and optionally a TCR or CAR is typicallyachieved by operably linking a nucleic acid encoding the chimericprotein and TCR/CAR polypeptide or portions thereof to one or morepromoters, and incorporating the construct into an expression vector.

Additional promoter elements, e.g., enhancers, regulate the frequency oftranscriptional initiation. Typically, these are located in the region30-110 bp upstream of the start site, although a number of promotershave recently been shown to contain functional elements downstream ofthe start site as well. The spacing between promoter elements frequentlyis flexible, so that promoter function is preserved when elements areinverted or moved relative to one another. In the thymidine kinase (tk)promoter, the spacing between promoter elements can be increased to 50bp apart before activity begins to decline.

One example of a suitable promoter is the immediate earlycytomegalovirus (CMV) promoter sequence. This promoter sequence is astrong constitutive promoter sequence capable of driving high levels ofexpression of any polynucleotide sequence operatively linked thereto.Another example of a suitable promoter is Elongation Growth Factor-1α(EF-1α). However, other constitutive promoter sequences may also beused, including, but not limited to the simian virus 40 (SV40) earlypromoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus(HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, MSCVpromoter, MND promoter, an avian leukemia virus promoter, anEpstein-Barr virus immediate early promoter, a Rous sarcoma viruspromoter, as well as human gene promoters such as, but not limited to,the actin promoter, the myosin promoter, the hemoglobin promoter, andthe creatine kinase promoter.

The vectors can be suitable for replication and integration ineukaryotic cells. Typical cloning vectors contain transcription andtranslation terminators, initiation sequences, and promoters useful forregulation of the expression of the desired nucleic acid sequence. Viralvector technology is well known in the art and is described, forexample, in Sambrook et al. (2001, Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory, New York), and in other virologyand molecular biology manuals, see also, WO 01/96584; WO 01/29058; andU.S. Pat. No. 6,326,193). In some embodiments, the constructs expressedare as shown in SEQ ID NOS:32-65 and 67-79. In some embodiments thenucleic acids are multi-cistronic constructs that permit the expressionof multiple transgenes (e.g., chimeric protein and a TCR and/or CARetc.) under the control of a single promoter. In some embodiments, thetransgenes (e.g., chimeric protein and a TCR and/or CAR etc.) areseparated by a self-cleaving 2A peptide. Examples of 2A peptides usefulin the nucleic acid constructs of the invention include F2A, P2A, T2Aand E2A. In other embodiments of the invention, the nucleic acidconstruct of the invention is a multi-cistronic construct comprising twopromoters; one promoter driving the expression of chimeric protein andthe other promoter driving the expression of the TCR or CAR. In someembodiments, the dual promoter constructs of the invention areuni-directional. In other embodiments, the dual promoter constructs ofthe invention are bi-directional. In order to assess the expression ofthe chimeric protein polypeptide or portions thereof, the expressionvector to be introduced into a cell can also contain either a selectablemarker gene or a reporter gene or both to facilitate identification andselection of expressing cells from the population of cells sought to betransfected or transduced through viral vectors.

Prior to expansion and genetic modification, a source of cells (e.g.,immune effector cells, e.g., T cells or NK cells) is obtained from asubject. The term “subject” is intended to include living organisms inwhich an immune response can be elicited (e.g., mammals). Examples ofsubjects include humans, dogs, cats, mice, rats, and transgenic speciesthereof. T cells can be obtained from a number of sources, includingperipheral blood mononuclear cells, bone marrow, lymph node tissue, cordblood, thymus tissue, tissue from a site of infection, ascites, pleuraleffusion, spleen tissue, and tumors.

In one aspect, T cells are isolated from peripheral blood lymphocytes bylysing the red blood cells and depleting the monocytes, for example, bycentrifugation through a PERCOLL™ gradient or by counterflow centrifugalelutriation.

In another aspect, Tumor infiltrating cells (TILs) are isolated and/orexpanded from a tumor, for example by a fragmented, dissected, or enzymedigested tumor biopsy or mass.

A specific subpopulation of T cells, such as CD3+, CD28+, CD4+, CD8+,CD45RA+, and CD45RO+T cells, can be further isolated by positive ornegative selection techniques. For example, in one aspect, T cells areisolated by incubation with anti-CD3/anti-CD28-conjugated beads, such asDYNABEADS® M-450 CD3/CD28 T, for a time period sufficient for positiveselection of the desired T cells. In one aspect, the time period isabout 30 minutes. In a further aspect, the time period ranges from 30minutes to 36 hours or longer and all integer values there between. In afurther aspect, the time period is at least 1, 2, 3, 4, 5, or 6 hours.In yet another preferred aspect, the time period is 10 to 24 hours. Inone aspect, the incubation time period is 24 hours. Longer incubationtimes may be used to isolate T cells in any situation where there arefew T cells as compared to other cell types, such in isolating tumorinfiltrating lymphocytes (TIL) from tumor tissue or fromimmunocompromised individuals. Further, use of longer incubation timescan increase the efficiency of capture of CD8+ T cells. Thus, by simplyshortening or lengthening the time T cells are allowed to bind to theCD3/CD28 beads and/or by increasing or decreasing the ratio of beads toT cells (as described further herein), subpopulations of T cells can bepreferentially selected for or against at culture initiation or at othertime points during the process. Additionally, by increasing ordecreasing the ratio of anti-CD3 and/or anti-CD28 antibodies on thebeads or other surface, subpopulations of T cells can be preferentiallyselected for or against at culture initiation or at other desired timepoints. The skilled artisan would recognize that multiple rounds ofselection can also be used in the context of this invention. In certainaspects, it may be desirable to perform the selection procedure anduse“the “unselected” cells in the activation and exp“nsion proc”ss.“Unselected” cells can also be subjected to further rounds of selection.

Enrichment of a T cell population by negative selection can beaccomplished with a combination of antibodies directed to surfacemarkers unique to the negatively selected cells. One method is cellsorting and/or selection via negative magnetic immunoadherence or flowcytometry that uses a cocktail of monoclonal antibodies directed to cellsurface markers present on the cells negatively selected. For example,to enrich for CD4+ cells by negative selection, a monoclonal antibodycocktail typically includes antibodies to CD14, CD20, CD16, HLA-DR, andCD8. In certain aspects, it may be desirable to enrich for or positivelyselect for regulatory T cells which typically express CD4+, CD25+,CD62Lhi, GITR+, CD137, PD1, TIM3, LAG-3, CD150 and FoxP3+.Alternatively, in certain aspects, T regulatory cells are depleted byanti-CD25 conjugated beads or other similar method of selection.

The methods described herein can include, e.g., selection of a specificsubpopulation of immune effector cells, e.g., T cells, that are a Tregulatory cell-depleted population, CD25+ depleted cells, using, e.g.,a negative selection technique, e.g., described herein. Preferably, thepopulation of T regulatory depleted cells contains less than 30%, 25%,20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% of CD25+ cells.

A specific subpopulation of chimeric protein effector cells thatspecifically bind to a target antigen can be enriched for by positiveselection techniques. For example, in some embodiments, effector cellsare enriched for by incubation with target antigen-conjugated beads fora time period sufficient for positive selection of the desired abTCReffector cells. In some embodiments, the time period is about 30minutes. In some embodiments, the time period ranges from 30 minutes to36 hours or longer (including all ranges between these values). In someembodiments, the time period is at least one, 2, 3, 4, 5, or 6 hours. Insome embodiments, the time period is 10 to 24 hours. In someembodiments, the incubation time period is 24 hours. For isolation ofeffector cells present at low levels in the heterogeneous cellpopulation, use of longer incubation times, such as 24 hours, canincrease cell yield. Longer incubation times may be used to isolateeffector cells in any situation where there are few effector cells ascompared to other cell types. The skilled artisan would recognize thatmultiple rounds of selection can also be used in the context of thisinvention.

T cells for stimulation can also be frozen after a washing step. Afterthe washing step that removes plasma and platelets, the cells may besuspended in a freezing solution. While many freezing solutions andparameters are known in the art and will be useful in this context, onemethod involves using PBS containing 20% DMSO and 8% human serumalbumin, or culture media containing 10% Dextran 40 and 5% Dextrose, 20%Human Serum Albumin and 7.5% DMSO, or 31.25% Plasmalyte-A, 31.25%Dextrose 5%, 0.45% NaCl, 10% Dextran 40 and 5% Dextrose, 20% Human SerumAlbumin, and 7.5% DMSO or other suitable cell freezing media containingfor example, Hespan and PlasmaLyte A, the cells then are frozen to −80°C. at a rate of 1° per minute and stored in the vapor phase of a liquidnitrogen storage tank. Other methods of controlled freezing may be usedas well as uncontrolled freezing immediately at −20° C. or in liquidnitrogen.

In embodiments described herein, the immune effector cell can be anallogeneic immune effector cell, e.g., T cell or NK cell.

A T cell described herein can be, e.g., engineered such that it does notexpress a functional HLA on its surface. For example, a T cell describedherein, can be engineered such that cell surface expression HLA, e.g.,HLA class 1 and/or HLA class II, is downregulated. In some aspects,downregulation of HLA may be accomplished by reducing or eliminatingexpression of beta-2 microglobulin (B2M).

In some embodiments, the T cell can lack a functional TCR and afunctional HLA, e.g., HLA class I and/or HLA class II. Modified T cellsthat lack expression of a functional TCR and/or HLA can be obtained byany suitable means, including a knock out or knock down of one or moresubunit of TCR or HLA. For example, the T cell can include a knock downof TCR and/or HLA using siRNA, shRNA, clustered regularly interspacedshort palindromic repeats (CRISPR) transcription-activator like effectornuclease (TALEN), or zinc finger endonuclease (ZFN).

In some embodiments, the allogeneic cell can be a cell which does notexpresses or expresses at low levels an inhibitory molecule, e.g. a cellengineered by any method described herein. For example, the cell can bea cell that does not express or expresses at low levels an inhibitorymolecule, e.g., that can decrease the ability of a chimericprotein-expressing cell to mount an immune effector response. Examplesof inhibitory molecules include PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM(e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAGS, VISTA, BTLA, TIGIT,LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM(TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, Gal9,adenosine, and TGFR beta. Inhibition of an inhibitory molecule, e.g., byinhibition at the DNA, RNA or protein level, can optimize aCAR-expressing cell performance. In embodiments, an inhibitory nucleicacid, e.g., an inhibitory nucleic acid, e.g., a dsRNA, e.g., an siRNA orshRNA, a clustered regularly interspaced short palindromic repeats(CRISPR), a transcription-activator like effector nuclease (TALEN), or azinc finger endonuclease (ZFN), e.g., as described herein, can be used.

T cells may be activated and expanded generally using methods asdescribed, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055;6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575;7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874;6,797,514; 6,867,041; and U.S. Patent Application Publication No.20060121005.

Generally, the T cells of the invention may be expanded by contact witha surface having attached thereto an agent that stimulates a CD3/TCRcomplex associated signal and a ligand that stimulates a costimulatorymolecule on the surface of the T cells. In particular, T cellpopulations may be stimulated as described herein, such as by contactwith an anti-CD3 antibody, or antigen-binding fragment thereof, or ananti-CD2 antibody immobilized on a surface, or by contact with a proteinkinase C activator (e.g., bryostatin) in conjunction with a calciumionophore. For co-stimulation of an accessory molecule on the surface ofthe T cells, a ligand that binds the accessory molecule is used. Forexample, a population of T cells can be contacted with an anti-CD3antibody and an anti-CD28 antibody, under conditions appropriate forstimulating proliferation of the T cells. To stimulate proliferation ofeither CD4+ T cells or CD8+ T cells, an anti-CD3 antibody and ananti-CD28 antibody can be used. Examples of an anti-CD28 antibodyinclude 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France) can be used ascan other methods commonly known in the art (Berg et al., TransplantProc. 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med.190(9):13191328, 1999; Garland et al., J. Immunol Meth. 227(1-2):53-63,1999).

In some embodiments, expansion can be performed using flasks orcontainers, or gas-permeable containers known by those of skill in theart and can proceed for 7 days, 8 days, 9 days, 10 days, 11 days, 12days, 13 days, or 14 days, about 7 days to about 14 days, about 8 daysto about 14 days, about 9 days to about 14 days, about 10 days to about14 days, about 11 days to about 14 days, about 12 days to about 14 days,or about 13 days to about 14 days. In some embodiments, the second TILexpansion can proceed for about 14 days.

In some embodiments, the expansion can be performed using non-specificT-cell receptor stimulation in the presence of interleukin-2 (IL-2) orinterleukin-15 (IL-15). The non-specific T-cell receptor stimulus caninclude, for example, an anti-CD3 antibody, such as about 30 ng/ml ofOKT3, a mouse monoclonal anti-CD3 antibody (commercially available fromOrtho-McNeil, Raritan, N.J. or Miltenyi Biotech, Auburn, Calif.) orUHCT-1 (commercially available from BioLegend, San Diego, Calif., USA).Chimeric protein cells can be expanded in vitro by including one or moreantigens, including antigenic portions thereof, such as epitope(s), of acancer, which can be optionally expressed from a vector, such as a humanleukocyte antigen A2 (HLA-A2) binding peptide, e.g., 0.3 .mu.MMART-1:26-35 (27L) or gp100:209-217 (210M), optionally in the presenceof a T-cell growth factor, such as 300 IU/mL IL-2 or IL-15. Othersuitable antigens may include, e.g., NY-ESO-1, TRP-1, TRP-2, tyrosinasecancer antigen, MAGE-A3, SSX-2, and VEGFR2, or antigenic portionsthereof. Chimeric protein cells may also be rapidly expanded byre-stimulation with the same antigen(s) of the cancer pulsed ontoHLA-A2-expressing antigen-presenting cells. Alternatively, the chimericprotein cells can be further stimulated with, e.g., example, irradiated,autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytesand IL-2. In some embodiments, the stimulation occurs as part of theexpansion. In some embodiments, the expansion occurs in the presence ofirradiated, autologous lymphocytes or with irradiated HLA-A2+ allogeneiclymphocytes and IL-2.

In some embodiments, the cell culture medium comprises IL-2. In someembodiments, the cell culture medium comprises about 1000 IU/mL, about1500 IU/mL, about 2000 IU/mL, about 2500 IU/mL, about 3000 IU/mL, about3500 IU/mL, about 4000 IU/mL, about 4500 IU/mL, about 5000 IU/mL, about5500 IU/mL, about 6000 IU/mL, about 6500 IU/mL, about 7000 IU/mL, about7500 IU/mL, or about 8000 IU/mL, or between 1000 and 2000 IU/mL, between2000 and 3000 IU/mL, between 3000 and 4000 IU/mL, between 4000 and 5000IU/mL, between 5000 and 6000 IU/mL, between 6000 and 7000 IU/mL, between7000 and 8000 IU/mL, or between 8000 IU/mL of IL-2.

In some embodiments, the cell culture medium comprises OKT3 antibody. Insome embodiments, the cell culture medium comprises about 0.1 ng/mL,about 0.5 ng/mL, about 1 ng/mL, about 2.5 ng/mL, about 5 ng/mL, about7.5 ng/mL, about 10 ng/mL, about 15 ng/mL, about 20 ng/mL, about 25ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 50 ng/mL,about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL, about100 ng/mL, about 200 ng/mL, about 500 ng/mL, about 1 μg/mL or between0.1 ng/mL and 1 ng/mL, between 1 ng/mL and 5 ng/mL, between 5 ng/mL and10 ng/mL, between 10 ng/mL and 20 ng/mL, between 20 ng/mL and 30 ng/mL,between 30 ng/mL and 40 ng/mL, between 40 ng/mL and 50 ng/mL, or between50 ng/mL and 100 ng/mL of OKT3 antibody.

In some embodiments, a combination of IL-2, IL-7, IL-15, and/or IL-21are employed as a combination during the expansion. In some embodiments,IL-2, IL-7, IL-15, and/or IL-21 as well as any combinations thereof canbe included during the expansion. In some embodiments, a combination ofIL-2, IL-15, and IL-21 are employed as a combination during theexpansion. In some embodiments, IL-2, IL-15, and IL-21 as well as anycombinations thereof can be included.

In some embodiments, the expansion can be conducted in a supplementedcell culture medium comprising IL-2, OKT-3, and antigen-presentingfeeder cells.

In some embodiments, the expansion culture media comprises about 500IU/mL of IL-15, about 400 IU/mL of IL-15, about 300 IU/mL of IL-15,about 200 IU/mL of IL-15, about 180 IU/mL of IL-15, about 160 IU/mL ofIL-15, about 140 IU/mL of IL-15, about 120 IU/mL of IL-15, or about 100IU/mL of IL-15, or about 500 IU/mL of IL-15 to about 100 IU/mL of IL-15,or about 400 IU/mL of IL-15 to about 100 IU/mL of IL-15 or about 300IU/mL of IL-15 to about 100 IU/mL of IL-15 or about 200 IU/mL of IL-15,or about 180 IU/mL of IL-15.

In some embodiments, the expansion culture media comprises about 20IU/mL of IL-21, about 15 IU/mL of IL-21, about 12 IU/mL of IL-21, about10 IU/mL of IL-21, about 5 IU/mL of IL-21, about 4 IU/mL of IL-21, about3 IU/mL of IL-21, about 2 IU/mL of IL-21, about 1 IU/mL of IL-21, orabout 0.5 IU/mL of IL-21, or about 20 IU/mL of IL-21 to about 0.5 IU/mLof IL-21, or about 15 IU/mL of IL-21 to about 0.5 IU/mL of IL-21, orabout 12 IU/mL of IL-21 to about 0.5 IU/mL of IL-21, or about 10 IU/mLof IL-21 to about 0.5 IU/mL of IL-21, or about 5 IU/mL of IL-21 to about1 IU/mL of IL-21, or about 2 IU/mL of IL-21. In some embodiments, thecell culture medium comprises about 1 IU/mL of IL-21, or about 0.5 IU/mLof IL-21.

In some embodiments the antigen-presenting feeder cells (APCs) arePBMCs. In an embodiment, the ratio of chimeric protein cells to PBMCsand/or antigen-presenting cells in the expansion is about 1 to 25, about1 to 50, about 1 to 100, about 1 to 125, about 1 to 150, about 1 to 175,about 1 to 200, about 1 to 225, about 1 to 250, about 1 to 275, about 1to 300, about 1 to 325, about 1 to 350, about 1 to 375, about 1 to 400,or about 1 to 500, or between 1 to 50 and 1 to 300, or between 1 to 100and 1 to 200.

In certain aspects, the primary stimulatory signal and the costimulatorysignal for the T cell may be provided by different protocols. Forexample, the agents providing each signal may be in solution or coupledto a surface. When coupled to a surface, the agents may be coupled tothe same su“fac” (i.e., in “cis” formation) or to separate sur“aces“i.e., in “trans” formation). Alternatively, one agent may be coupled toa surface and the other agent in solution. In one aspect, the agentproviding the costimulatory signal is bound to a cell surface and theagent providing the primary activation signal is in solution or coupledto a surface. In certain aspects, both agents can be in solution. In oneaspect, the agents may be in soluble form, and then cross-linked to asurface, such as a cell expressing Fc receptors or an antibody or otherbinding agent which will bind to the agents. In this regard, see forexample, U.S. Patent Application Publication Nos. 20040101519 and20060034810 for artificial antigen presenting cells (aAPCs) that arecontemplated for use in activating and expanding T cells in the presentinvention.

In one aspect, the two agents are immobilized on beads, either on thesame bead, i.e., “cis,” or to separate beads, i.e., “trans.” By way ofexample, the agent providing the primary activation signal is ananti-CD3 antibody or an antigen-binding fragment thereof and the agentproviding the costimulatory signal is an anti-CD28 antibody orantigen-binding fragment thereof; and both agents are co-immobilized tothe same bead in equivalent molecular amounts. In one aspect, a 1:1ratio of each antibody bound to the beads for CD4+ T cell expansion andT cell growth is used. In certain aspects of the present invention, aratio of anti CD3:CD28 antibodies bound to the beads is used such thatan increase in T cell expansion is observed as compared to the expansionobserved using a ratio of 1:1. In one particular aspect an increase offrom about 1 to about 3 fold is observed as compared to the expansionobserved using a ratio of 1:1. In one aspect, the ratio of CD3:CD28antibody bound to the beads ranges from 100:1 to 1:100 and all integervalues there between. In one aspect of the present invention, moreanti-CD28 antibody is bound to the particles than anti-CD3 antibody,i.e., the ratio of CD3:CD28 is less than one. In certain aspects of theinvention, the ratio of anti CD28 antibody to anti CD3 antibody bound tothe beads is greater than 2:1. In one particular aspect, a 1:100CD3:CD28 ratio of antibody bound to beads is used. In one aspect, a 1:75CD3:CD28 ratio of antibody bound to beads is used. In a further aspect,a 1:50 CD3:CD28 ratio of antibody bound to beads is used. In one aspect,a 1:30 CD3:CD28 ratio of antibody bound to beads is used. In onepreferred aspect, a 1:10 CD3:CD28 ratio of antibody bound to beads isused. In one aspect, a 1:3 CD3:CD28 ratio of antibody bound to the beadsis used. In yet one aspect, a 3:1 CD3:CD28 ratio of antibody bound tothe beads is used.

Ratios of particles to cells from 1:500 to 500:1 and any integer valuesin between may be used to stimulate T cells or other target cells. Asthose of ordinary skill in the art can readily appreciate, the ratio ofparticles to cells may depend on particle size relative to the targetcell. For example, small sized beads could only bind a few cells, whilelarger beads could bind many. In certain aspects the ratio of cells toparticles ranges from 1:100 to 100:1 and any integer values in-betweenand in further aspects the ratio comprises 1:9 to 9:1 and any integervalues in between, can also be used to stimulate T cells. The ratio ofanti-CD3- and anti-CD28-coupled particles to T cells that result in Tcell stimulation can vary as noted above, however certain preferredvalues include 1:100, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8, 1:7, 1:6,1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1,and 15:1 with one preferred ratio being at least 1:1 particles per Tcell. In one aspect, a ratio of particles to cells of 1:1 or less isused. In one particular aspect, a preferred particle:cell ratio is 1:5.In further aspects, the ratio of particles to cells can be varieddepending on the day of stimulation. For example, in one aspect, theratio of particles to cells is from 1:1 to 10:1 on the first day andadditional particles are added to the cells every day or every other daythereafter for up to 10 days, at final ratios of from 1:1 to 1:10 (basedon cell counts on the day of addition). In one particular aspect, theratio of particles to cells is 1:1 on the first day of stimulation andadjusted to 1:5 on the third and fifth days of stimulation. In oneaspect, particles are added on a daily or every other day basis to afinal ratio of 1:1 on the first day, and 1:5 on the third and fifth daysof stimulation. In one aspect, the ratio of particles to cells is 2:1 onthe first day of stimulation and adjusted to 1:10 on the third and fifthdays of stimulation. In one aspect, particles are added on a daily orevery other day basis to a final ratio of 1:1 on the first day, and 1:10on the third and fifth days of stimulation. One of skill in the art willappreciate that a variety of other ratios may be suitable for use in thepresent invention. In particular, ratios will vary depending on particlesize and on cell size and type. In one aspect, the most typical ratiosfor use are in the neighborhood of 1:1, 2:1 and 3:1 on the first day.

In further aspects of the present invention, the cells, such as T cells,are combined with agent-coated beads, the beads and the cells aresubsequently separated, and then the cells are cultured. In analternative aspect, prior to culture, the agent-coated beads and cellsare not separated but are cultured together. In a further aspect, thebeads and cells are first concentrated by application of a force, suchas a magnetic force, resulting in increased ligation of cell surfacemarkers, thereby inducing cell stimulation.

Viral- and non-viral-based genetic engineering tools can be used togenerate chimeric protein cells, including without limitation T cells,NK cells resulting in permanent or transient expression of therapeuticgenes. Retrovirus-based gene delivery is a mature, well-characterizedtechnology, which has been used to permanently integrate CARs into thehost cell genome (Scholler J., e.g. Decade-long safety and function ofretroviral-modified chimeric antigen receptor T cells. Sci. Transl. Med.2012; 4:132ra53; Rosenberg S. A. et al., Gene transfer intohumans—immunotherapy of patients with advanced melanoma, usingtumor-infiltrating lymphocytes modified by retroviral gene transduction.N. Engl. J. Med. 1990; 323:570-578)

Non-viral DNA transfection methods can also be used. For example, Singhet al describes use of the Sleeping Beauty (SB) transposon systemdeveloped to engineer CAR T cells (Singh H., et al., Redirectingspecificity of T-cell populations for CD19 using the Sleeping Beautysystem. Cancer Res. 2008; 68:2961-2971) and is being used in clinicaltrials (see e.g., ClinicalTrials.gov: NCT00968760 and NCT01653717). Thesame technology is applicable to engineer chimeric protein cells.

Multiple SB enzymes have been used to deliver transgenes. Matesdescribes a hyperactive transposase (SB100X) with approximately 100-foldenhancement in efficiency when compared to the first-generationtransposase. SB100X supported 35-50% stable gene transfer in humanCD34(+) cells enriched in hematopoietic stem or progenitor cells. (MatesL. et al., Molecular evolution of a novel hyperactive Sleeping Beautytransposase enables robust stable gene transfer in vertebrates. Nat.Genet. 2009; 41:753-761) and multiple transgenes can be delivered frommulticistronic single plasmids (e.g., Thokala R. et al., Redirectingspecificity of T cells using the Sleeping Beauty system to expresschimeric antigen receptors by mix-and-matching of VL and VH domainstargeting cD123+ tumors. PLoS ONE. 2016; 11:e0159477) or multipleplasmids (e.g., Hurton L. V. et al., Tethered IL-15 augments antitumoractivity and promotes a stem-cell memory subset in tumor-specific Tcells. Proc. Natl. Acad. Sci. USA. 2016; 113:E7788-E7797). Such systemsare used with chimeric proteins of the invention.

Morita et al, describes the piggyBac transposon system to integratelarger transgenes (Morita D. et al., Enhanced expression of anti-CD19chimeric antigen receptor in piggyBac transposon-engineered T cells.Mol. Ther. Methods Clin. Dev. 2017; 8:131-140) Nakazawa et al. describesuse of the system to generate EBV-specific cytotoxic T-cells expressingHER2-specific chimeric antigen receptor (Nakazawa Y et al,PiggyBac-mediated cancer immunotherapy using EBV-specific cytotoxicT-cells expressing HER2-specific chimeric antigen receptor. Mol. Ther.2011; 19:2133-2143). Manuri et al used the system to generate CD-19specific T cells (Manuri P. V. R. et al., piggyBactransposon/transposase system to generate CD19-specific T cells for thetreatment of B-lineage malignancies. Hum. Gene Ther. 2010; 21:427-437).

Transposon technology is easy and economical. One potential drawback isthe longer expansion protocols currently employed may result in T celldifferentiation, impaired activity and poor persistence of the infusedcells. Monjezi et al describe development minicircle vectors thatminimize these difficulties through higher efficiency integrations(Monjezi R. et al., Enhanced CAR T-cell engineering using non-viralSleeping Beauty transposition from minicircle vectors. Leukemia. 2017;31:186-194). These transposon technologies can be used for chimericproteins of the invention.

The present invention also relates to a pharmaceutical compositioncontaining a vector or a chimeric protein expressing cell of theinvention together with a pharmaceutically acceptable carrier, diluentor excipient, and optionally one or more further pharmaceutically activepolypeptides and/or compounds.

In some embodiments, a pharmaceutical composition is provided comprisinga chimeric protein described above and a pharmaceutically acceptablecarrier. In some embodiments, a pharmaceutical composition is providedcomprising a nucleic acid encoding a chimeric protein according to anyof the embodiments described above and a pharmaceutically acceptablecarrier. In some embodiments, a pharmaceutical composition is providedcomprising an effector cell expressing a chimeric protein describedabove and a pharmaceutically acceptable carrier. Such a formulation may,for example, be in a form suitable for intravenous infusion.

As used herein, by “pharmaceutically acceptable” or “pharmacologicallycompatible” is meant a material that is not biologically or otherwiseundesirable, e.g., the material may be incorporated into apharmaceutical composition administered to a patient without causing anysignificant undesirable biological effects or interacting in adeleterious manner with any of the other components of the compositionin which it is contained. Pharmaceutically acceptable carriers orexcipients have preferably met the required standards of toxicologicaland manufacturing testing and/or are included on the Inactive IngredientGuide prepared by the U.S. Food and Drug administration.

An aspect of the invention provides a population of modified T cellsexpressing a recombinant chimeric protein. A suitable population may beproduced by a method described above.

The population of modified T cells may be for use as a medicament. Forexample, a population of modified T cells as described herein may beused in cancer immunotherapy therapy, for example adoptive T celltherapy.

Other aspects of the invention provide the use of a population ofmodified T cells as described herein for the manufacture of a medicamentfor the treatment of cancer, a population of modified T cells asdescribed herein for the treatment of cancer, and a method of treatmentof cancer may comprise administering a population of modified T cells asdescribed herein to an individual in need thereof.

The population of modified T cells may be autologous i.e. the modified Tcells were originally obtained from the same individual to whom they aresubsequently administered (i.e. the donor and recipient individual arethe same). A suitable population of modified T cells for administrationto the individual may be produced by a method comprising providing aninitial population of T cells obtained from the individual, modifyingthe T cells to express a cAMP PDE or fragment thereof and an antigenreceptor which binds specifically to cancer cells in the individual, andculturing the modified T cells.

The population of modified T cells may be allogeneic i.e. the modified Tcells were originally obtained from a different individual to theindividual to whom they are subsequently administered (i.e. the donorand recipient individual are different). The donor and recipientindividuals may be HLA matched to avoid GVHD and other undesirableimmune effects. A suitable population of modified T cells foradministration to a recipient individual may be produced by a methodcomprising providing an initial population of T cells obtained from adonor individual, modifying the T cells to express a chimeric proteinwhich binds specifically to cancer cells in the recipient individual,and culturing the modified T cells.

Following administration of the modified T cells, the recipientindividual may exhibit a T cell mediated immune response against cancercells in the recipient individual. This may have a beneficial effect onthe cancer condition in the individual.

Cancer conditions may be characterized by the abnormal proliferation ofmalignant cancer cells and may include leukaemias, such as AML, CML, ALLand CLL, lymphomas, such as Hodgkin lymphoma, non-Hodgkin lymphoma andmultiple myeloma, and solid cancers such as sarcomas, skin cancer,melanoma, bladder cancer, brain cancer, breast cancer, uterus cancer,ovary cancer, prostate cancer, lung cancer, colorectal cancer, cervicalcancer, liver cancer, head and neck cancer, oesophageal cancer, pancreascancer, renal cancer, adrenal cancer, stomach cancer, testicular cancer,cancer of the gall bladder and biliary tracts, thyroid cancer, thymuscancer, cancer of bone, and cerebral cancer, as well as cancer ofunknown primary (CUP).

Cancer cells within an individual may be immunologically distinct fromnormal somatic cells in the individual (i.e. the cancerous tumor may beimmunogenic). For example, the cancer cells may be capable of elicitinga systemic immune response in the individual against one or moreantigens expressed by the cancer cells. The tumor antigens that elicitthe immune response may be specific to cancer cells or may be shared byone or more normal cells in the individual.

An individual suitable for treatment as described above may be a mammal,such as a rodent (e.g. a guinea pig, a hamster, a rat, a mouse), murine(e.g. a mouse), canine (e.g. a dog), feline (e.g. a cat), equine (e.g. ahorse), a primate, simian (e.g. a monkey or ape), a monkey (e.g.marmoset, baboon), an ape (e.g. gorilla, chimpanzee, orangutan, gibbon),or a human.

In preferred embodiments, the individual is a human. In other preferredembodiments, non-human mammals, especially mammals that areconventionally used as models for demonstrating therapeutic efficacy inhumans (e.g. murine, primate, porcine, canine, or rabbit animals) may beemployed.

The term “therapeutically effective amount” refers to an amount of achimeric protein or composition comprising a chimeric protein asdisclosed herei“, eff”ctive to “treat” a disease or disorder in anindividual. In the case of cancer, the therapeutically effective amountof a chimeric protein or composition comprising a chimeric protein asdisclosed herein can reduce the number of cancer cells; reduce the tumorsize or weight; inhibit (i.e., slow to some extent and preferably stop)cancer cell infiltration into peripheral organs; inhibit (i.e., slow tosome extent and preferably stop) tumor metastasis; inhibit, to someextent, tumor growth; and/or relieve to some extent one or more of thesymptoms associated with the cancer. To the extent a chimeric protein orcomposition comprising a chimeric protein as disclosed herein canprevent growth and/or kill existing cancer cells, it can be cytostaticand/or cytotoxic. In some embodiments, the therapeutically effectiveamount is a growth inhibitory amount. In some embodiments, thetherapeutically effective amount is an amount that improves progressionfree survival of a patient. In the case of infectious disease, such asviral infection, the therapeutically effective amount of a chimericprotein or composition comprising a chimeric protein as disclosed hereincan reduce the number of cells infected by the pathogen; reduce theproduction or release of pathogen-derived antigens; inhibit (i.e., slowto some extent and preferably stop) spread of the pathogen to uninfectedcells; and/or relieve to some extent one or more symptoms associatedwith the infection. In some embodiments, the therapeutically effectiveamount is an amount that extends the survival of a patient.

Cells, including T and NK cells, expressing chimeric proteins for use inthe methods of the present may either be created ex vivo eithe' from apatient's own peripheral blood (autologous), or in the setting of ahaematopoietic stem cell transplant from donor peripheral blood(allogenic), or peripheral blood from an unconnected donor (allogenic).Alternatively, T-cells or NK cells may be derived from ex-vivodifferentiation of inducible progenitor cells or embryonic progenitorcells to T-cells or NK cells. In these instances, T-cells expressing achimeric protein and, optionally, a CAR and/or TCR, are generated byintroducing DNA or RNA coding for the chimeric protein and, optionally,a CAR and/or TCR, by one of many means including transduction with aviral vector, transfection with DNA or RNA.

T or NK cells expressing a chimeric protein of the present inventionand, optionally, expressing a TCR and/or CAR may be used for thetreatment of haematological cancers or solid tumors.

A method for the treatment of disease relates to the therapeutic use ofa vector or cell, including a T or NK cell, of the invention. In thisrespect, the vector, or T or NK cell may be administered to a subjecthaving an existing disease or condition in order to lessen, reduce orimprove at least one symptom associated with the disease and/or to slowdown, reduce or block the progression of the disease. The method of theinvention may cause or promote T-cell mediated killing of cancer cells.The vector, or T or NK cell according to the present invention may beadministered to a patient with one or more additional therapeuticagents. The one or more additional therapeutic agents can beco-administered to the patient. By “co-administering” is meantadministering one or more additional therapeutic agents and the vector,or T or NK cell of the present invention sufficiently close in time suchthat the vector, or T or NK cell can enhance the effect of one or moreadditional therapeutic agents, or vice versa. In this regard, thevectors or cells can be administered first and the one or moreadditional therapeutic agents can be administered second, or vice versa.Alternatively, the vectors or cells and the one or more additionaltherapeutic agents can be administered simultaneously. Oneco-administered therapeutic agent that may be useful is IL-2, as this iscurrently used in existing cell therapies to boost the activity ofadministered cells. However, IL-2 treatment is associated with toxicityand tolerability issues.

In some embodiments, the addition of the engineered protein to a subjectinduces cytokine secretion. In some embodiments, the addition of CoStARto a subject induces cytokine secretion. In some embodiments, thecytokine secretion lowers cytokine levels in the subject, including butnot limited to IL-2. In some embodiments, the cytokine secretionfollowing CoStAR exposure results in no detectable IL-2 in the subject.In some embodiments, the addition of the engineered protein to a subjectreduces or eliminates the need for administration of exogenous IL-2. Insome embodiments, the addition of the CoStAR to a subject reduces oreliminates the need for administration of exogenous IL-2.

In some embodiments, other mechanisms of action are involved in thekilling of tumor cells apart from the direct effect of CoStAR. In someembodiments, secretion of cytokines and/or proliferation are evaluated.In some embodiments, tumor cell killing potency is characterized by flowcytometry to enumerate T cells and target cells and plate-basedfluorescence or luminescence to measure percent killing. In someembodiments, cytokine secretion potency is characterized at the singlecell level by flow cytometry and ELISA/MSD to characterize thepopulation. In some embodiments, proliferation potency is determined byflow cytometry to characterize the population. In some embodiments, TILpotency may be determined by additional analytes, memory phenotype,cytotoxicity using cell lines, cytotoxicity using a patient specifictumor, a cytokine panel, cell proliferation and/or cellular composition.

As mentioned, for administration to a patient, the chimeric proteineffector cells can be allogeneic or autologous to the patient. In someembodiments, allogeneic cells are further genetically modified, forexample by gene editing, so as to minimize or prevent GVHD and/or apatient's immune response against the chimeric protein cells.

The chimeric protein effector cells are used to treat cancers andneoplastic diseases associated with a target antigen. Cancers andneoplastic diseases that may be treated using any of the methodsdescribed herein include tumors that are not vascularized, or not yetsubstantially vascularized, as well as vascularized tumors. The cancersmay comprise non-solid tumors (such as hematological tumors, forexample, leukemias and lymphomas) or may comprise solid tumors. Types ofcancers to be treated with the chimeric protein effector cells of theinvention include, but are not limited to, carcinoma, blastoma, andsarcoma, and certain leukemia or lymphoid malignancies, benign andmalignant tumors, and malignancies e.g., sarcomas, carcinomas, andmelanomas. Adult tumors/cancers and pediatric tumors/cancers are alsoincluded.

Hematologic cancers are cancers of the blood or bone marrow. Examples ofhematological (or hematogenous) cancers include leukemias, includingacute leukemias (such as acute lymphocytic leukemia, acute myelocyticleukemia, acute myelogenous leukemia and myeloblastic, promyelocytic,myelomonocytic, monocytic and erythroleukemia), chronic leukemias (suchas chronic myelocytic (granulocytic) leukemia, chronic myelogenousleukemia, and chronic lymphocytic leukemia), polycythemia vera,l'mphoma, Hodgkin's dise'se, non-Hodgkin's lymphoma (indolent and highgrade forms), multiple myeloma, plasmacyt'ma, Waldenstrom'smacroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairycell leukemia and myelodysplasia.

Solid tumors are abnormal masses of tissue that usually do not containcysts or liquid areas. Solid tumors can be benign or malignant.Different types of solid tumors are named for the type of cells thatform them (such as sarcomas, carcinomas, and lymphomas). Examples ofsolid tumors, such as sarcomas and carcinomas, include adrenocorticalcarcinoma, cholangiocarcinoma, fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteosarcoma, and other sarcomas, synovioma,mes'thelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, coloncarcinoma, stomach cancer, lymphoid malignancy, pancreatic cancer,breast cancer, lung cancers, ovarian cancer, prostate cancer,hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma,adenocarcinoma, sweat gland carcinoma, thyroid cancer (e.g., medullarythyroid carcinoma and papillary thyroid carcinoma), pheochromocytomassebaceous gland carcinoma, papillary carcinoma, papillaryadenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cellcarcinoma, hepatoma, bile duct carcinoma, chorio'arcinoma, Wilms' tumor,cervical cancer (e.g., cervical carcinoma and pre-invasive cervicaldysplasia), colorectal cancer, cancer of the anus, anal canal, oranorectum, vaginal cancer, cancer of the vulva (e.g., squamous cellcarcinoma, intraepithelial carcinoma, adenocarcinoma, and fibrosarcoma),penile cancer, oropharyngeal cancer, esophageal cancer, head cancers(e.g., squamous cell carcinoma), neck cancers (e.g., squamous cellcarcinoma), testicular cancer (e.g., seminoma, teratoma, embryonalcarcinoma, teratocarcinoma, choriocarcinoma, sarcoma, Leydig cell tumor,fibroma, fibroadenoma, adenomatoid tumors, and lipoma), bladdercarcinoma, kidney cancer, melanoma, cancer of the uterus (e.g.,endometrial carcinoma), urothelial cancers (e.g., squamous cellcarcinoma, transitional cell carcinoma, adenocarcinoma, ureter cancer,and urinary bladder cancer), and CNS tumors (such as a glioma (such asbrainstem glioma and mixed gliomas), glioblastoma (also known asglioblastoma multiforme) astrocytoma, CNS lymphoma, germinoma,medulloblastoma, Schwannoma craniopharyogioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma,neuroblastoma, retinoblastoma and brain metastases).

When “an immunologically effective amount,” “an anti-tumor effectiveamount,” “a tumor-inhibiting effective amount,” or “therapeutic amount”is indicated, the precise amount of the compositions of the presentinvention to be administered can be determined by a physician withconsideration of individual differences in age, weight, tumor size,extent of infection or metastasis, and condition of the patient(subject). It can generally be stated that a pharmaceutical compositioncomprising the T cells described herein may be administered at a dosageof 10⁴ to 10⁹ cells/kg body weight, in some instances 10⁵ to 10⁶cells/kg body weight, including all integer values within those ranges.T cell compositions may also be administered multiple times at thesedosages. The cells can be administered by using infusion techniques thatare commonly known in immunotherapy (see, e.g., Rosenberg et al., NewEng. J. of Med. 319:1676, 1988).

A chimeric protein-expressing cell described herein may be used incombination with other known agents and therapie. “Administered” “incombination”, as used herein, means that two (or more) differenttreatments are delivered to the subject during the cours' of thesubject's affliction with the disorder, e.g., the two or more treatmentsare delivered after the subject has been diagnosed with the disorder andbefore the disorder has been cured or eliminated or treatment has ceasedfor other reasons. In some embodiments, the delivery of one treatment isstill occurring when the delivery of the second begins, so that there isoverlap in terms of administration. This is sometimes referr“d toherein”s “s“multaneous” or “con”urrent delivery”. In other embodiments,the delivery of one treatment ends before the delivery of the othertreatment begins. In some embodiments of either case, the treatment ismore effective because of combined administration. For example, thesecond treatment is more effective, e.g., an equivalent effect is seenwith less of the second treatment, or the second treatment reducessymptoms to a greater extent, than would be seen if the second treatmentwere administered in the absence of the first treatment, or theanalogous situation is seen with the first treatment. In someembodiments, delivery is such that the reduction in a symptom, or otherparameter related to the disorder is greater than what would be observedwith one treatment delivered in the absence of the other. The effect ofthe two treatments can be partially additive, wholly additive, orgreater than additive. The delivery can be such that an effect of thefirst treatment delivered is still detectable when the second isdelivered.

A chimeric protein-expressing cell described herein and the at least oneadditional therapeutic agent can be administered simultaneously, in thesame or in separate compositions, or sequentially. For sequentialadministration, the CAR-expressing cell described herein can beadministered first, and the additional agent can be administered second,or the order of administration can be reversed.

The chimeric protein therapy and/or other therapeutic agents, proceduresor modalities can be administered during periods of active disorder, orduring a period of remission or less active disease. The chimericprotein therapy can be administered before the other treatment,concurrently with the treatment, post-treatment, or during remission ofthe disorder.

When administered in combination, the therapy and the additional agent(e.g., second or third agent), or all, can be administered in an amountor dose that is higher, lower or the same than the amount or dosage ofeach agent used individually, e.g., as a monotherapy. In someembodiments, the administered amount or dosage of the chimeric proteintherapy, the additional agent (e.g., second or third agent), or all, islower (e.g., at least 20%, at least 30%, at least 40%, or at least 50%)than the amount or dosage of each agent used individually, e.g., as amonotherapy. In other embodiments, the amount or dosage of the chimericprotein therapy, the additional agent (e.g., second or third agent), orall, that results in a desired effect (e.g., treatment of cancer) islower (e.g., at least 20%, at least 30%, at least 40%, or at least 50%lower) than the amount or dosage of each agent used individually, e.g.,as a monotherapy, required to achieve the same therapeutic effect.

In further aspects, a chimeric protein-expressing cell described hereinmay be used in a treatment regimen in combination with surgery,chemotherapy, radiation, immunosuppressive agents, such as cyclosporin,azathioprine, methotrexate, mycophenolate, and FK506, antibodies, orother immunoablative agents such as CAMPATH, anti-CD3 antibodies orother antibody therapies, cytoxin, fludarabine, cyclosporin, FK506,rapamycin, mycophenolic acid, steroids, FR901228, cytokines, andirradiation, peptide vaccine, such as that described in Izumoto et al.2008 J Neurosurg 108:963-971.

In certain instances, compounds of the present invention are combinedwith other therapeutic agents, such as other anti-cancer agents,anti-allergic agents, anti-nausea agents (or anti-emetics), painrelievers, cytoprotective agents, and combinations thereof.

In one embodiment, a chimeric protein-expressing cell described hereincan be used in combination with a chemotherapeutic agent. Exemplarychemotherapeutic agents include an anthracycline (e.g., doxorubicin(e.g., liposomal doxorubicin)), a vinca alkaloid (e.g., vinblastine,vincristine, vindesine, vinorelbine), an alkylating agent (e.g.,cy53acarbazineide, decarbazine, melphalan, ifosfamide, temozolomide), animmune cell antibody (e.g., alemtuzamab, gemtuzumab, rituximab,ofatumumab, tositumomab, brentuximab), an antimetabolite (including,e.g., folic acid antagonists, pyrimidine analogs, purine analogs andadenosine deaminase inhibitors (e.g., fludarabine)), an mTOR inhibitor,a TNFR glucocorticoid induced TNFR related protein (GITR) agonist, aproteasome inhibitor (e.g., aclacinomycin A, gliotoxin or bortezomib),an immunomodulator such as thalidomide or a thalidomide derivative(e.g., lenalidomide).

General Chemotherapeutic agents considered for use in combinationtherapies include busulfan (Myleran®), busulfan injection (Busulfex®),cladribine (Leustatin®), cyclophosphamide (Cytoxan® or Neosar®),cytarabine, cytosine arabinoside (Cytosar-U®), cytarabine liposomeinjection (DepoCyt®), daunorubicin hydrochloride (Cerubidine®),daunorubicin citrate liposome injection (DaunoXome®), dexamethasone,doxorubicin hydrochloride (Adriamycin®, Rubex®), etoposide (Vepesid®),fludarabine phosphate (Fludara®), hydroxyurea (Hydrea®), Idarubicin(Idamycin®), mitoxantrone (Novantrone®), Gemtuzumab Ozogamicin(Mylotarg®).

In embodiments, general chemotherapeutic agents considered for use incombination therapies include anastrozole (Arimidex®), bicalutamide(Casodex®), bleomycin sulfate (Blenoxane®), busulfan (Myleran®),busulfan injection (Busulfex®), capecitabine (Xeloda®),N4-pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin (Paraplatin®),carmustine (BiCNU®), chlorambucil (Leukeran®), cisplatin (Platinol®),cladribine (Leustatin®), cyclophosphamide (Cytoxan® or Neosar®),cytarabine, cytosine arabinoside (Cytosar-U®), cytarabine liposomeinjection (DepoCyt®), dacarbazine (DTIC-Dome®), dactinomycin(Actinomycin D, Cosmegan), daunorubicin hydrochloride (Cerubidine®),daunorubicin citrate liposome injection (DaunoXome®), dexamethasone,docetaxel (Taxotere®), doxorubicin hydrochloride (Adriamycin®, Rubex®),etoposide (Vepesid®), fludarabine phosphate (Fludara®), 5-fluorouracil(Adrucil®, Efudex®), flutamide (Eulexin®), tezacitibine, Gemcitabine(difluorodeoxycitidine), hydroxyurea (Hydrea®), Idarubicin (Idamycin®),ifosfamide (IFEX®), irinotecan (Camptosar®), L-asparaginase (ELSPAR®),leucovorin calcium, melphalan (Alkeran®), 6-mercaptopurine(Purinethol®), methotrexate (Folex®), mitoxantrone (Novantrone®),mylotarg, paclitaxel (Taxol®), phoenix (Yttrium90/MX-DTPA), pentostatin,polifeprosan 20 with carmustine implant (Gliadel®), tamoxifen citrate(Nolvadex®), teniposide (Vumon®), 6-thioguanine, thiotepa, tirapazamine(Tirazone®), topotecan hydrochloride for injection (Hycamptin®),vinblastine (Velban®), vincristine (Oncovin®), and vinorelbine(Navelbine®).

Treatments can be evaluated, for example, by tumor regression, tumorweight or size shrinkage, time to progression, duration of survival,progression free survival, overall response rate, duration of response,quality of life, protein expression and/or activity. Approaches todetermining efficacy of the therapy can be employed, including forexample, measurement of response through radiological imaging.

TCR Incorporated Antigen Agnostic Receptors (TIAARs)

Table 2 provides exemplary, non-limiting examples of components of TCRincorporated antigen agnostic receptors (TIAARs) of the invention. Table3 shows exemplary arrangements of the components.

TABLE 2 TCR incorporated antigen agnostic receptor (TIAAR) componentsCode Signal peptide Tag ECD_TMD ICD Costim pIB1026 CD3D Myc CD3D N/ACD28-CD40 pIB1027 CD3E FLAG CD3E N/A CD28-CD40 pIB1028 CD3G Myc CD3G N/ACD28-CD40 pIB1029 CD3Z Myc IC CD3Z N/A CD28-CD40 pIB1030 CD8A Myc hTRDCN/A CD28-CD40 pIB1031 CD8A FLAG hTRGC1 N/A CD28-CD40 pIB1032 CD8A MycmTRAC N/A CD28-CD40 pIB1033 CD8A FLAG mTRBC1 N/A CD28-CD40 pIB1046 CD8A×2 Myc and hTRDC_hTRGC1 N/A CD28-CD40 FLAG pIB1047 CD8A ×2 Myc andmTRAC_mTRBC1 N/A CD28-CD40 FLAG pIB1048 CD3D and Myc and CD3D_CD3E N/ACD28-CD40 CD3E FLAG pIB1049 CD3G and Myc and CD3G_CD3E N/A CD28-CD40CD3E FLAG pIB1050 CD3D and Myc and CD3D_CD3E CD3D_CD3E CD28-CD40 CD3EFLAG pIB1051 CD3D and Myc and CD3D_CD3E CD3D_CD3E N/A CD3E FLAG pIB1052CD3D and Myc and CD3D_CD3E N/A N/A CD3E FLAG pIB1053 CD3G and Myc andCD3G_CD3E CD3G_CD3E CD28-CD40 CD3E FLAG pIB1054 CD3G and Myc andCD3G_CD3E CD3G_CD3E N/A CD3E FLAG pIB1055 CD3G and Myc and CD3G_CD3E N/AN/A CD3E FLAG pIB1056 CD3Z Myc IC CD3Z CD3Z CD28-CD40 pIB1057 CD3Z MycIC CD3Z CD3Z N/A pIB1058 CD3Z Myc IC CD3Z N/A N/A pIB1059 CD3Z Myc ICCD3Z CD3Z (×2) CD28-CD40 pIB1060 CD3Z Myc IC CD3Z CD3Z (×2) N/A pIB1061CD3Z Myc IC CD3Z CD3Z (×2) CD28-CD40 (swaped) pIB1062 CD3Z Myc IC CD3ZCD3Z CD28-CD40 (swaped) pIB1063 CD80 Myc CD80 CD80 N/A pIB1064 no signalMyc IC N/A Lck N/A peptide pIB1065 no signal Myc IC N/A Lck (Y505F) N/Apeptide pIB1066 CD80 Myc CD80 Lck N/A pIB1067 CD80 Myc CD80 LckCD28-CD40 pIB1068 CD80 Myc CD80 CD80_Lck N/A (Y505F) pIB1069 CD80 MycCD80 CD80_Lck CD28-CD40 (Y505F) pIB1070 CD8A Myc LAT LAT N/A pIB1071CD8A Myc LAT LAT CD28-CD40 pIB1072 CD4 Myc CD4 CD4 CD28-CD40 pIB1073 CD4Myc CD4 CD4 CD28-CD40 pIB1074 CD8A and Myc and CD8A and CD8A and N/ACD8B FLAG CD8B CD8B pIB1075 CD8A and Myc and CD8A and CD8A and CD28-CD40CD8B FLAG CD8B CD8B

TABLE 3 TCR incorporated antigen agnostic receptors (TIAARs) CodeDescription pIB1026 CD3D_CD3D_CD28CD40 pIB1027 CD3E_CD3E_CD28CD40pIB1028 CD3G_CD3G_CD28CD40 pIB1029 CD3Z_CD3Z_CD28CD40_Myc pIB1030CD8A_hTRDC_CD28CD40 pIB1031 CD8A_hTRGC1_CD28CD40 pIB1032CD8A_mTRAC_CD28CD40 pIB1033 CD8A_mTRBC1_CD28CD40 pIB1046CD8A_hTRDC_CD28CD40-T2A-CD8a_hTRGC1_CD28CD40 pIB1047CD8A_mTRAC_CD28CD40-T2A-CD8A_mTRBC1_CD28CD40 pIB1048CD3D_CD3D_CD28CD40-T2A-CD3E_CD3E_CD28CD40 pIB1049CD3G_CD3G_CD28CD40-T2A-CD3E_CD3E_CD28CD40 pIB1050 CD3D_CD3D_CD3D(ICD)_CD28CD40-T2A-CD3E_CD3E_CD3E (ICD)_CD28CD40 pIB1051 CD3D_CD3D_CD3DICD -T2A-CD3E_CD3E_CD3E ICD pIB1052 CD3D_CD3D (control)-T2A-CD3E_CD3E(control) pIB1053 CD3G_CD3G_CD3G (ICD)_CD28CD40-T2A-CD3E_CD3E_CD3E(ICD)_CD28CD40 pIB1054 CD3G_CD3G_CD3G ICD -T2A-CD3E_CD3E_CD3E ICDpIB1055 CD3G_CD3G (control)-T2A-CD3E_CD3E (control) pIB1056CD3z_CD3z_CD3z ICD_CD28CD40_Myc pIB1057 CD3z_CD3z_CD3z ICD_Myc pIB1058CD3z_CD3z (control) pIB1059 CD3Z_CD3Z_CD3Z ICD (duplicating CD3zendodomain-6 ITAMs)_CD28CD40_Myc pIB1060 CD3Z_CD3Z_CD3Z ICD (duplicatingCD3z endodomain-6 ITAMs)_Myc pIB1061 CD3Z_CD3Z_CD28CD40_CD3Z (6ITAMs)_Myc pIB1062 CD3Z_CD3Z_CD28CD40_CD3Z ICD_Myc pIB1063 CD80(control) pIB1064 Lck (control) pIB1065 Lck (Y505F) (control) pIB1066CD80_Lck pIB1067 CD80_Lck_CD28CD40 pIB1068 CD80_Lck (Y505F) pIB1069CD80_Lck (Y505F)_CD28CD40 pIB1070 LAT (control) pIB1071 LAT_C28CD40pIB1072 CD4 control pIB1073 CD4_CD28_CD40 pIB1074 CD8 control pIB1075CD8_CD28_CD40

Constitutive Costimulatory Proteins

Table 4 provides exemplary, non-limiting examples of components ofconstitutive costimulatory proteins of the invention. Table 5 shows theexemplary arrangements of the components.

TABLE 4 Constitutively stimulating antigen agnostic receptor (C-SAAR)components Code Signal peptide Tag ECD_TMD Costim pIB1076 CD8A Myc LZ(cFos)_EGFR CD28-CD40 pIB1077 CD8A Myc LZ (cFos)_CD28 CD28-CD40 pIB1078CD8A Myc LZ (cJun)_EGFR CD28-CD40 pIB1079 CD8A Myc LZ (cJun)_CD28CD28-CD40 pIB1080 CD8A Myc LZ (c/EBP)_EGFR CD28-CD40 pIB1081 CD8A Myc LZ(c/EBP)_CD28 CD28-CD40 pIB1103 GpA Myc GpA ECD_TMD CD28-CD40 pIB1104 GpAMyc GpA TMD CD28-CD40 pIB1105 EPOR Myc EPOR ECD_TMD CD28-CD40 pIB1106EPOR Myc EPOR TMD CD28-CD40 pIB1107 TPOR Myc TPOR ECD_TMD CD28-CD40pIB1108 TPOR Myc TPOR TMD CD28-CD40 pIB1109 TPOR Myc TPOR ECD_TMD(S505N) CD28-CD40 pIB1110 TPOR Myc TPOR TMD (S505N) CD28-CD40 pIB1111TPOR Myc TPOR ECD_TMD (W515K) CD28-CD40 pIB1112 TPOR Myc TPOR TMD(W515K) CD28-CD40 pIB1113 TPOR Myc TPOR ECD_TMD (H499L) CD28-CD40pIB1114 TPOR Myc TPOR TMD (H499L) CD28-CD40 pIB1115 TPOR Myc TPORECD_TMD (S505N-W515K) CD28-CD40 pIB1116 TPOR Myc TPOR TMD (S505N-W515K)CD28-CD40 pIB1117 TPOR Myc TPOR ECD_TMD (H499Y-S505N) CD28-CD40 pIB1118TPOR Myc TPOR TMD (H499Y-S505N) CD28-CD40 pIB1119 TPOR Myc TPOR ECD_TMD(L498W-H499C) CD28-CD40 pIB1120 TPOR Myc TPOR TMD (L498W-H499C)CD28-CD40 pIB1025 CD8a Myc CD28 CD28-CD40 pIB1179 CD8a N/A IgG1 + CD28TMCD28-CD40 pIB1180 CD8a N/A IgG1mut + CD28TM CD28-CD40 pIB1181 CD8a N/AIgG2 + CD28TM CD28-CD40 pIB1182 CD8a N/A IgG3 + CD28TM CD28-CD40 pIB1183CD8a N/A IgG4 + CD28TM CD28-CD40 pIB1184 CD8a N/A IgG4mut +CD28TMCD28-CD40 pIB1185 CD8a N/A IgG1 + CD28 stalk/TM CD28-CD40 pIB1186 CD8aN/A IgG1mut + CD28 stalk/TM CD28-CD40 pIB1187 CD8a N/A IgG2 + CD28stalk/TM CD28-CD40 pIB1188 CD8a N/A IgG3 + CD28 stalk/TM CD28-CD40pIB1189 CD8a N/A IgG4 + CD28 stalk/TM CD28-CD40 pIB1190 CD8a N/AIgG4mut + CD28 stalk/TM CD28-CD40

TABLE 5 C-SAAR Proteins Code Description pIB1076 LZ (cFos)- EGFRTM/JMD-CD28-CD40 pIB1077 LZ (cFos)- CD28TM- CD28-CD40 pIB1078 LZ (cJun)-EGFRTM/JMD- CD28-CD40 pIB1079 LZ (cJun)- CD28TM- CD28-CD40 pIB1080 LZ(c/EBP)- EGFRTM/JMD- CD28-CD40 pIB1081 LZ (c/EBP)- CD28TM- CD28-CD40pIB1103 GpA ECD-TMD-CD28-CD40 pIB1104 GpA TMD-CD28-CD40 pIB1105 EpoRECD-TMD-CD28-CD40 pIB1106 EpoR TMD-CD28-CD40 pIB1107 TPO ECD- TPO (WT)TMD -CD28-CD40 pIB1108 TPO (WT) TMD -CD28-CD40 pIB1109 TPO ECD- TPO(S505N) TMD -CD28-CD40 pIB1110 TPO (S505N) TMD -CD28-CD40 pIB1111 TPOECD-TPO (W515K) TMD -CD28-CD40 pIB1112 TPO (W515K) TMD -CD28-CD40pIB1113 TPO ECD- TPO (H499L) TMD -CD28-CD40 pIB1114 TPO (H499L) TMD-CD28-CD40 pIB1115 TPO ECD- TPO (S505N-W515K) TMD -CD28-CD40 pIB1116 TPO(S505N-W515K) TMD -CD28-CD40 pIB1117 TPO ECD- TPO (H499Y-S505N) TMD-CD28-CD40 pIB1118 TPO (H499Y-S505N) TMD -CD28-CD40 pIB1119 TPO ECD- TPO(L498W-H499C) TMD -CD28-CD40 pIB1120 TPO (L498W-H499C) TMD -CD28-CD40pIB1025 CD28 TM_CD28_CD40 pIB1179IICH2CH3)-CD28(TM)-CD28(CoStim)-CD40(CoStim) pIB1180 IgG1ICH3,mutant)-CD28(TM)-CD28(CoStim)-CD40(CoStim) pIB1182IgG3(CH2CH3)-CD28(TM)-CD28(CoStim)-CD40(CoI) pIB1184 IgG4(CH2CH3,mutant)-CD28(TM)-CD28(CoStim)-CD40(CoStim) pIB1185IgG1(CH2CH3)-CD28(Stalk + TM)-CD28(CoStim)-CD40(CoStim) pIB1186IgG1(CH2CH3, mutant)-CD28(Stalk + TM)-CD28(CoStim)-CD40(CoStim) pIB1187IgG2(CH2CH3)-CD28(Stalk + TM)-CD28(CoStim)-CD40(CoStim) pIB1188IgG3(CH2CH3)-CD28(Stalk + TM)-CD28(CoStim)-CD40(CoStim) pIB1189IgG4(CH2CH3)-CD28(Stalk + TM)-CD28(CoStim)-CD40(CoStim) pIB1190IgG4(CH2CH3, mutant)-CD28(Stalk + TM)-CD28(CoStim)-CD40(CoStim)

Inducible Costimulatory Receptors

Table 6 provides exemplary, non-limiting examples of induciblecostimulatory receptors of the invention. Table 7 shows exemplaryarrangements of the components.

TABLE 6 Inducible costimulatory protein components Code Signal peptideTag ECD_TMD ICD Costim pIB1082 EGFR Myc EGFR N/A CD28-CD40 pIB1083 EGFRMyc EGFR (domain IV) N/A CD28-CD40 pIB1084 EGFR Myc EGFR (623-668) N/ACD28-CD40 pIB1085 Her2 Myc Her2 N/A CD28-CD40 pIB1086 Her2 Myc Her2(V659E) N/A CD28-CD40 pIB1087 Her2 Myc Her2 (V660D) N/A CD28-CD40pIB1088 Her2 Myc Her2 (V660R) N/A CD28-CD40 pIB1089 Her2 Myc Her2 domainIV_TMD N/A CD28-CD40 pIB1090 Her2 Myc Her2 domain IV_TMD (V659E) N/ACD28-CD40 pIB1091 Her2 Myc Her2 domain IV_TMD (G660D) N/A CD28-CD40pIB1092 Her2 Myc Her2 domain IV_TMD (G660R) N/A CD28-CD40 pIB1093 Her2Myc Her2 TMD N/A CD28-CD40 pIB1094 Her2 Myc Her2 TMD (V659E) N/ACD28-CD40 pIB1095 Her2 Myc Her2 TMD (G660D) N/A CD28-CD40 pIB1096 Her2Myc Her2 TMD (G660R) N/A CD28-CD40 pIB1097 CD8A Myc A30514 VH_VL N/ACD28-CD40 pIB1098 CD8A Myc A30514 VL_VH N/A CD28-CD40 pIB1099 CD8A MycA30523 VH_VL N/A CD28-CD40 pIB1100 CD8A Myc A30523 VL_VH N/A CD28-CD40pIB1101 CD8A Myc A30633 VH_VL N/A CD28-CD40 pIB1102 CD8A Myc A30633VL_VH N/A CD28-CD40

TABLE 7 Inducible costimulatory proteins Code GOI description pIB1082 WTEGFR ECD- EGFRTM/JMD- CD28-CD40 pIB1083 domain IV - EGFRTM/JMD-CD28-CD40 pIB1084 EGFRTM/JMD- CD28-CD40 (control) pIB1085 Her 2 (Domain1 to IV)-TMD/JMD-CD28-CD40 pIB1086 Her 2 (Domain 1 to IV)-TMD(V659E)/JMD-CD28-CD40 pIB1087 Her 2 (Domain 1 to IV)-TMD(G660D)/JMD-CD28-CD40 pIB1088 Her 2 (Domain 1 to IV)-TMD(G660R)/JMD-CD28-CD40 pIB1089 Her 2 (Domain IV)-TMD/JMD-CD28-CD40pIB1090 Her 2 (Domain IV)-TMD (V659E)/JMD-CD28-CD40 pIB1091 Her 2(Domain IV)-TMD (G660D)/JMD-CD28-CD40 pIB1092 Her 2 (Domain IV)-TMD(G660R)/JMD-CD28-CD40 pIB1093 Her 2 TMD/JMD-CD28-CD40 pIB1094 Her 2 TMD(V659E)/JMD-CD28-CD40 pIB1095 Her 2 TMD (G660D)/JMD-CD28-CD40 pIB1096Her 2 TMD (G660R)/JMD-CD28-CD40 pIB1097 Anti-ID1 VH-VL(A30514-pembrolizumab)- CD28TMD CD28-CD40 pIB1098 Anti-ID1 VL-VH(A30514-pembrolizumab)- CD28TMD CD28-CD40 pIB1099 Anti-ID2 Vh-VL(A30523-pembrolizumab)- CD28TMD CD28-CD40 pIB1100 Anti-ID2 VL-Vh(A30523-pembrolizumab)- CD28TMD CD28-CD40 pIB1101 Anti-ID3 Vh-VL(A30633-pembrolizumab)- CD28TMD CD28-CD40 pIB1102 Anti-ID3 VL-VH(A30633-pembrolizumab)- CD28TMD CD28-CD40

The following sequences in the below table include complete componentsand are non-limiting. Components may include a signal peptide (SP), aTCR clustering domain (CD) and/or a signaling domain (SD). It will beunderstood that whereas certain proteins may comprise N-terminal signalpeptides when expressed, those signal peptides are cleaved and may beimprecisely cleaved when the proteins are expressed, and that theresulting proteins from which signal peptides are removed comprisebinding domains having variation of up to about five amino acids in thelocation of the N-terminal amino acid.

TABLE 8 TCR costimulation construct examples SEQ ID NO: 1 Component:SP CD3D_ Sequence: MEHSTFLSGL VLATLLSQVS P SEQ ID NO: 2 Component:CD CD3D_ Sequence:FKIPIEELED RVFVNCNTSI TWVEGTVGTL LSDITRLDLG KRILDPRGIYRCNGTDIYKD KESTVQVHYR MCQSCVELDP ATVAGIIVTD VIATLLLALG VFCFASEQ ID NO: 3 Component: SD CD28CD40 Sequence:RSKRSRLLHS DYMNMTPRRP GPTRKHYQPY APPRDFAAYR SKKVAKKPTNKAPHPKQEPQ EINFPDDLPG SNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ SEQ ID NO: 4Full length: CD3D_CD3D_CD28CD40 Sequence:MEHSTFLSGL VLATLLSQVS PFKIPIEELE DRVFVNCNTS ITWVEGTVGTLLSDITRLDL GKRILDPRGI YRCNGTDIYK DKESTVQVHY RMCQSCVELDPATVAGIIVT DVIATLLLAL GVFCFARSKR SRLLHSDYMN MTPRRPGPTRKHYQPYAPPR DFAAYRSKKV AKKPTNKAPH PKQEPQEINF PDDLPGSNTAAPVQETLHGC QPVTQEDGKE SRISVQERQ SEQ ID NO: 5 Component: SP CD3E_Sequence: MQSGTHWRVL GLCLLSVGVW GQ SEQ ID NO: 6 Component: CD CD3E_Sequence: DGNEEMGGIT QTPYKVSISG TTVILTCPQY PGSEILWQHN DKNIGGDEDDKNIGSDEDHL SLKEFSELEQ SGYYVCYPRG SKPEDANFYL YLRARVCENCMEMDVMSVAT IVIVDICITG GLLLLVYYWS SEQ ID NO: 7 Component: SD CD28CD40Sequence: RSKRSRLLHS DYMNMTPRRP GPTRKHYQPY APPRDFAAYR SKKVAKKPTNKAPHPKQEPQ EINFPDDLPG SNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ SEQ ID NO: 8Full length: CD3E_CD3E_CD28CD40 Sequence:MQSGTHWRVL GLCLLSVGVW GQDGNEEMGG ITQTPYKVSI SGTTVILTCPQYPGSEILWQH NDKNIGGDED DKNIGSDEDH LSLKEFSELE QSGYYVCYPRGSKPEDANFYL YLRARVCENC MEMDVMSVAT IVIVDICITG GLLLLVYYWSRSKRSRLLHSD YMNMTPRRPG PTRKHYQPYA PPRDFAAYRS KKVAKKPTNKAPHPKQEPQEI NFPDDLPGSN TAAPVQETLH GCQPVTQEDG KESRISVQERQ SEQ ID NO: 9Component: SP CD3G_ Sequence: MEQGKGLAVL ILAIILLQGT LA SEQ ID NO: 10Component: CD CD3G_ Sequence:QSIKGNHLVK VYDYQEDGSV LLTCDAEAKN ITWFKDGKMI GFLTEDKKKWNLGSNAKDPR GMYQCKGSQN KSKPLQVYYR MCQNCIELNA ATISGFLFAE IVSIFVLAVG VYFIASEQ ID NO: 11 Component: SD CD28CD40 Sequence:RSKRSRLLHS DYMNMTPRRP GPTRKHYQPY APPRDFAAYR SKKVAKKPTNKAPHPKQEPQ EINFPDDLPG SNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ SEQ ID NO: 12Full length: CD3G_CD3G_CD28CD40 Sequence:MEQGKGLAVL ILAIILLQGT LAQSIKGNHL VKVYDYQEDG SVLLTCDAEAKNITWFKDGK MIGFLTEDKK KWNLGSNAKD PRGMYQCKGS QNKSKPLQVYYRMCQNCIEL NAATISGFLF AEIVSIFVLA VGVYFIARSK RSRLLHSDYMNMTPRRPGPT RKHYQPYAPP RDFAAYRSKK VAKKPTNKAP HPKQEPQEINFPDDLPGSNT AAPVQETLHG CQPVTQEDGK ESRISVQERQ SEQ ID NO: 13 Component:SP CD3Z_ Sequence: MKWKALFTAA ILQAQLPITE A SEQ ID NO: 14 Component:CD CD3Z_ Sequence: QSFGLLDPKL CYLLDGILFI YGVILTALFL SEQ ID NO: 15Component: SD CD28CD40 Sequence:RSKRSRLLHS DYMNMTPRRP GPTRKHYQPY APPRDFAAYR SKKVAKKPTNKAPHPKQEPQ EINFPDDLPG SNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ SEQ ID NO: 16Full length: CD3Z_CD3Z_CD28CD40 Sequence:MKWKALFTAA ILQAQLPITE AQSFGLLDPK LCYLLDGILF IYGVILTALFLRSKRSRLLH SDYMNMTPRR PGPTRKHYQP YAPPRDFAAY RSKKVAKKPTNKAPHPKQEP QEINFPDDLP GSNTAAPVQE TLHGCQPVTQ EDGKESRISV QERQSEQ ID NO: 17 Component: SP CD8A_ Sequence: MALPVTALLL PLALLLHAAR PSEQ ID NO: 18 Component: CD TRDC_ Sequence:SQPHTKPSVF VMKNGTNVAC LVKEFYPKDI RINLVSSKKI TEFDPAIVISPSGKYNAVKL GKYEDSNSVT CSVQHDNKTV HSTDFEVKTD STDHVKPKETENTKQPSKSC HKPKAIVHTE KVNMMSLTVL GLRMLFAKTV AVNFLLTAKL FFL SEQ ID NO: 19Component: SD CD28CD40 Sequence:RSKRSRLLHS DYMNMTPRRP GPTRKHYQPY APPRDFAAYR SKKVAKKPTNKAPHPKQEPQ EINFPDDLPG SNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ SEQ ID NO: 20Full length: CD8A_TRDC_CD28CD40 Sequence:MALPVTALLL PLALLLHAAR PSQPHTKPSV FVMKNGTNVA CLVKEFYPKDIRINLVSSKK ITEFDPAIVI SPSGKYNAVK LGKYEDSNSV TCSVQHDNKTVHSTDFEVKT DSTDHVKPKE TENTKQPSKS CHKPKAIVHT EKVNMMSLTVLGLRMLFAKT VAVNFLLTAK LFFLRSKRSR LLHSDYMNMT PRRPGPTRKHYQPYAPPRDF AAYRSKKVAK KPTNKAPHPK QEPQEINFPD DLPGSNTAAPVQETLHGCQP VTQEDGKESR ISVQERQ SEQ ID NO: 21 Component: SP CD8A_Sequence: MALPVTALLL PLALLLHAAR P SEQ ID NO: 22 Component: CD TRGC1_Sequence: DKQLDADVSP KPTIFLPSIA ETKLQKAGTY LCLLEKFFPD VIKIHWQEKKSNTILGSQEG NTMKTNDTYM KFSWLTVPEK SLDKEHRCIV RHENNKNGVDQEIIFPPIKT DVITMDPKDN CSKDANDTLL LQLTNTSAYY MYLLLLLKSV VYFAIITCCL LSEQ ID NO: 23 Component: SD CD28CD40 Sequence:RSKRSRLLHS DYMNMTPRRP GPTRKHYQPY APPRDFAAYR SKKVAKKPTNKAPHPKQEPQ EINFPDDLPG SNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ SEQ ID NO: 24Full length: CD8A_TRGC1_CD28CD40 Sequence:MALPVTALLL PLALLLHAAR PDKQLDADVS PKPTIFLPSI AETKLQKAGTYLCLLEKFFP DVIKIHWQEK KSNTILGSQE GNTMKTNDTY MKFSWLTVPEKSLDKEHRCI VRHENNKNGV DQEIIFPPIK TDVITMDPKD NCSKDANDTLLLQLTNTSAY YMYLLLLLKS VVYFAIITCC LLRSKRSRLL HSDYMNMTPRRPGPTRKHYQ PYAPPRDFAA YRSKKVAKKP TNKAPHPKQE PQEINFPDDLPGSNTAAPVQ ETLHGCQPVT QEDGKESRIS VQERQ SEQ ID NO: 25 Component: SP CD8A_Sequence: MALPVTALLL PLALLLHAAR P SEQ ID NO: 26 Component: CD TRAC_Sequence: IQNPEPAVYQ LKDPRSQDST LCLFTDFDSQ INVPKTMESG TFITDKCVLDMKAMDSKSNG AIAWSNQTSF TCQDIFKETN ATYPSSDVPC DATLTEKSFETDMNLNFQNL LVIVLRILLL KVAGFNLLMT LRLWSS SEQ ID NO: 27 Component:SD CD28CD40 Sequence:RSKRSRLLHS DYMNMTPRRP GPTRKHYQPY APPRDFAAYR SKKVAKKPTNKAPH PKQEP QEINFPDDLP GSNTAAPVQE TLHGCQPVTQ EDGKESRISV QERQSEQ ID NO: 28 Full length: CD8A_TRAC_CD28CD40 Sequence:MALPVTALLL PLALLLHAAR PIQNPEPAVY QLKDPRSQDS TLCLFTDFDSQINVPKTMES GTFITDKCVL DMKAMDSKSN GAIAWSNQTS FTCQDIFKETNATYPSSDVP CDATLTEKSF ETDMNLNFQN LLVIVLRILL LKVAGFNLLMTLRLWSSRSK RSRLLHSDYM NMTPRRPGPT RKHYQPYAPP RDFAAYRSKKVAKKPTNKAP HPKQEPQEIN FPDDLPGSNT AAPVQETLHG CQPVTQEDGK ESRISVQERQSEQ ID NO: 29 Component: SP CD8A_ Sequence: MALPVTALLL PLALLLHAAR PSEQ ID NO: 30 Component: CD TRBC1_ Sequence:VLTPPKVSLF EPSKAEIANK QKATLVCLAR GFFPDHVELS WWVNGKEVHSGVCTDPQAYK ESNYSYCLSS RLRVSATFWH NPRNHFRCQV QFHGLSEEDKWPEGSPKPVT QNISAEAWGR ADCGITSASY QQGVLSATIL YEILLGKATLYAVLVSTLVV MAMVKRKNS SEQ ID NO: 31 Component: SD CD28CD40 Sequence:RSKRSRLLHS DYMNMTPRRP GPTRKHYQPY APPRDFAAYR SKKVAKKPTNKAPHPKQEPQ EINFPDDLPG SNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ SEQ ID NO: 32Full length: CD8A_TRBC1_CD28CD40 Sequence:MALPVTALLL PLALLLHAAR PVLTPPKVSL FEPSKAEIAN KQKATLVCLARGFFPDHVEL SWWVNGKEVH SGVCTDPQAY KESNYSYCLS SRLRVSATFWHNPRNHFRCQ VQFHGLSEED KWPEGSPKPV TQNISAEAWG RADCGITSASYQQGVLSATI LYEILLGKAT LYAVLVSTLV VMAMVKRKNS RSKRSRLLHSDYMNMTPRRP GPTRKHYQPY APPRDFAAYR SKKVAKKPTN KAPHPKQEPQEINFPDDLPG SNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ SEQ ID NO: 33vector clone: pIB1001 SequenceMALPVTALLL PLALLLHAAR PEQKLISEED LQVQLVQSGA EVKKPGASVKVSCKASGYTF TSYWMNWVRQ APGQGLEWMG RIDPYDSETH YAQKLQGRVTMTTDTSTSTA YMELRSLRSD DTAVYYCARG GYDFDVGTLY WFFDVWGQGTTVTVSSGGGG SGGGGSGGGG SDIQMTQSPS SLSASVGDRV TITCRASENIYSYLAWYQQK PGKAPKLLIY NAKTLAEGVP SRFSGSGSGT DFTLTISSLQPEDFATYYCQ HHYGTPRTFG GGTKVEIKAA AGSGGSGILV KQSPMLVAYDNAVNLSCKYS YNLFSREFRA SLHKGLDSAV EVCVVYGNYS QQLQVYSKTGFNCDGKLGNE SVTFYLQNLY VNQTDIYFCK IEVMYPPPYL DNEKSNGTIIHVKGKHLCPS PLFPGPSKPF WVLVVVGGVL ACYSLLVTVA FIIFWVRSKRSRLLHSDYMN MTPRRPGPTR KHYQPYAPPR DFAAYRSKKV AKKPTNKAPHPKQEPQEINF PDDLPGSNTA APVQETLHGC QPVTQEDGKE SRISVQERQ SEQ ID NO: 34vector clone: pIB1002 Sequence:MALPVTALLL PLALLLHAAR PEQKLISEED LDIQMTQSPS SLSASVGDRVTITCRASENI YSYLAWYQQK PGKAPKLLIY NAKTLAEGVP SRFSGSGSGTDFTLTISSLQ PEDFATYYCQ HHYGTPRTFG GGTKVEIKGG GGSGGGGSGGGGSQVQLVQS GAEVKKPGAS VKVSCKASGY TFTSYWMNWV RQAPGQGLEWMGRIDPYDSE THYAQKLQGR VTMTTDTSTS TAYMELRSLR SDDTAVYYCARGGYDFDVGT LYWFFDVWGQ GTTVTVSSAA AGSGGSGILV KQSPMLVAYDNAVNLSCKYS YNLFSREFRA SLHKGLDSAV EVCVVYGNYS QQLQVYSKTGFNCDGKLGNE SVTFYLQNLY VNQTDIYFCK IEVMYPPPYL DNEKSNGTIIHVKGKHLCPS PLFPGPSKPF WVLVVVGGVL ACYSLLVTVA FIIFWVRSKRSRLLHSDYMN MTPRRPGPTR KHYQPYAPPR DFAAYRSKKV AKKPTNKAPHPKQEPQEINF PDDLPGSNTA APVQETLHGC QPVTQEDGKE SRISVQERQ SEQ ID NO: 35vector clone: pIB1003 Sequence:MALPVTALLL PLALLLHAAR PEQKLISEED LQVQLVESGG GVVQPGRSLRLSCAASGFTF SSYDMHWVRQ APGKGLEWVA VIWYDGSNKY YADSVKGRFTISRDNSKNTL YLQMNSLRAE DTAVYYCARG SGNWGFFDYW GQGTLVTVSSGGGGSGGGGS GGGGSDIQMT QSPSSLSASV GDRVTITCRA SQGISRWLAWYQQKPEKAPK SLIYAASSLQ SGVPSRFSGS GSGTDFTLTI SSLQPEDFATYYCQQYNTYP RTFGQGTKVE IKAAAGSGGS GILVKQSPML VAYDNAVNLSCKYSYNLFSR EFRASLHKGL DSAVEVCVVY GNYSQQLQVY SKTGFNCDGKLGNESVTFYL QNLYVNQTDI YFCKIEVMYP PPYLDNEKSN GTIIHVKGKHLCPSPLFPGP SKPFWVLVVV GGVLACYSLL VTVAFIIFWV RSKRSRLLHSDYMNMTPRRP GPTRKHYQPY APPRDFAAYR SKKVAKKPTN KAPHPKQEPQEINFPDDLPG SNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ SEQ ID NO: 36vector clone: pIB1004 Sequence:MALPVTALLL PLALLLHAAR PEQKLISEED LDIQMTQSPS SLSASVGDRVTITCRASQGI SRWLAWYQQK PEKAPKSLIY AASSLQSGVP SRFSGSGSGTDFTLTISSLQ PEDFATYYCQ QYNTYPRTFG QGTKVEIKGG GGSGGGGSGGGGSQVQLVES GGGVVQPGRS LRLSCAASGF TFSSYDMHWV RQAPGKGLEWVAVIWYDGSN KYYADSVKGR FTISRDNSKN TLYLQMNSLR AEDTAVYYCARGSGNWGFFD YWGQGTLVTV SSAAAGSGGS GILVKQSPML VAYDNAVNLSCKYSYNLFSR EFRASLHKGL DSAVEVCVVY GNYSQQLQVY SKTGFNCDGKLGNESVTFYL QNLYVNQTDI YFCKIEVMYP PPYLDNEKSN GTIIHVKGKHLCPSPLFPGP SKPFWVLVVV GGVLACYSLL VTVAFIIFWV RSKRSRLLHSDYMNMTPRRP GPTRKHYQPY APPRDFAAYR SKKVAKKPTN KAPHPKQEPQEINFPDDLPG SNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ SEQ ID NO: 37vector clone: pIB1005 Sequence:MALPVTALLL PLALLLHAAR PEQKLISEED LQVQLQQWGA GLLKPSETLSLTCAVYGGSF SGYYWSWIRQ SPEGLEWIGE INHGGYVTYN PSLESRVTISVDTSKNQFSL KLSSVTAADT AVYYCARDYG PGNYDWYFDL WGRGTLVTVSSGGGGSGGGG SGGGGSEIVL TQSPATLSLS PGERATLSCR ASQSVSSYLAWYQQKPGQAP RLLIYDASNR ATGIPARFSG SGSGTDFTLT ISSLEPEDFAVYYCQQRSNW PPALTFGGGT KVEIKRAAAG SGGSGILVKQ SPMLVAYDNAVNLSCKYSYN LFSREFRASL HKGLDSAVEV CVVYGNYSQQ LQVYSKTGFNCDGKLGNESV TFYLQNLYVN QTDIYFCKIE VMYPPPYLDN EKSNGTIIHVKGKHLCPSPL FPGPSKPFWV LVVVGGVLAC YSLLVTVAFI IFWVRSKRSRLLHSDYMNMT PRRPGPTRKH YQPYAPPRDF AAYRSKKVAK KPTNKAPHPKQEPQEINFPD DLPGSNTAAP VQETLHGCQP VTQEDGKESR ISVQERQ SEQ ID NO: 38vector clone : pIB1006 Sequence:MALPVTALLL PLALLLHAAR PEQKLISEED LEIVLTQSPA TLSLSPGERATLSCRASQSV SSYLAWYQQK PGQAPRLLIY DASNRATGIP ARFSGSGSGTDFTLTISSLE PEDFAVYYCQ QRSNWPPALT FGGGTKVEIK RGGGGSGGGGSGGGGSQVQL QQWGAGLLKP SETLSLTCAV YGGSFSGYYW SWIRQSPEGLEWIGEINHGG YVTYNPSLES RVTISVDTSK NQFSLKLSSV TAADTAVYYCARDYGPGNYD WYFDLWGRGT LVTVSSAAAG SGGSGILVKQ SPMLVAYDNAVNLSCKYSYN LFSREFRASL HKGLDSAVEV CVVYGNYSQQ LQVYSKTGFNCDGKLGNESV TFYLQNLYVN QTDIYFCKIE VMYPPPYLDN EKSNGTIIHVKGKHLCPSPL FPGPSKPFWV LVVVGGVLAC YSLLVTVAFI IFWVRSKRSRLLHSDYMNMT PRRPGPTRKH YQPYAPPRDF AAYRSKKVAK KPTNKAPHPKQEPQEINFPD DLPGSNTAAP VQETLHGCQP VTQEDGKESR ISVQERQ SEQ ID NO: 39vector clone: pIB1007 Sequence:MALPVTALLL PLALLLHAAR PEQKLISEED LQVTLRESGP ALVKPTQTLTLTCTFSGFSL STSGMGVGWI RQPPGKALEW LAHIWWDDDK YYNPSLKSRLTISKDTSKNQ VVLTMTNMDP VDTATYYCAR TRRYFPFAYW GQGTLVTVSSGGGGSGGGGS GGGGSEIVMT QSPATLSVSP GERATLSCKA SQNVGTNVAWYQQKPGQAPR LLIYSASYRY SGIPARFSGS GSGTEFTLTI SSLQSEDFAVYYCQQYNTDP LTFGGGTKVE IKAAAGSGGS GILVKQSPML VAYDNAVNLSCKYSYNLFSR EFRASLHKGL DSAVEVCVVY GNYSQQLQVY SKTGFNCDGKLGNESVTFYL QNLYVNQTDI YFCKIEVMYP PPYLDNEKSN GTIIHVKGKHLCPSPLFPGP SKPFWVLVVV GGVLACYSLL VTVAFIIFWV RSKRSRLLHSDYMNMTPRRP GPTRKHYQPY APPRDFAAYR SKKVAKKPTN KAPHPKQEPQEINFPDDLPG SNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ SEQ ID NO: 40vector clone: pIB1008 Sequence:MALPVTALLL PLALLLHAAR PEQKLISEED LEIVMTQSPA TLSVSPGERATLSCKASQNV GTNVAWYQQK PGQAPRLLIY SASYRYSGIP ARFSGSGSGTEFTLTISSLQ SEDFAVYYCQ QYNTDPLTFG GGTKVEIKGG GGSGGGGSGGGGSQVTLRES GPALVKPTQT LTLTCTFSGF SLSTSGMGVG WIRQPPGKALEWLAHIWWDD DKYYNPSLKS RLTISKDTSK NQVVLTMTNM DPVDTATYYCARTRRYFPFA YWGQGTLVTV SSAAAGSGGS GILVKQSPML VAYDNAVNLSCKYSYNLFSR EFRASLHKGL DSAVEVCVVY GNYSQQLQVY SKTGFNCDGKLGNESVTFYL QNLYVNQTDI YFCKIEVMYP PPYLDNEKSN GTIIHVKGKHLCPSPLFPGP SKPFWVLVVV GGVLACYSLL VTVAFIIFWV RSKRSRLLHSDYMNMTPRRP GPTRKHYQPY APPRDFAAYR SKKVAKKPTN KAPHPKQEPQEINFPDDLPG SNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ SEQ ID NO: 41vector clone: pIB1009 Sequence:MALPVTALLL PLALLLHAAR PEQKLISEED LQVQLVQSGV EVKKPGASVKVSCKASGYTF TNYYMYWVRQ APGQGLEWMG GINPSNGGTN FNEKFKNRVTLTTDSSTTTA YMELKSLQFD DTAVYYCARR DYRFDMGFDY WGQGTTVTVSSGGGGSGGGG SGGGGSEIVL TQSPATLSLS PGERATLSCR ASKGVSTSGYSYLHWYQQKP GQAPRLLIYL ASYLESGVPA RFSGSGSGTD FTLTISSLEPEDFAVYYCQH SRDLPLTFGG GTKVEIKRAA AGSGGSGILV KQSPMLVAYDNAVNLSCKYS YNLFSREFRA SLHKGLDSAV EVCVVYGNYS QQLQVYSKTGFNCDGKLGNE SVTFYLQNLY VNQTDIYFCK IEVMYPPPYL DNEKSNGTIIHVKGKHLCPS PLFPGPSKPF WVLVVVGGVL ACYSLLVTVA FIIFWVRSKRSRLLHSDYMN MTPRRPGPTR KHYQPYAPPR DFAAYRSKKV AKKPTNKAPHPKQEPQEINF PDDLPGSNTA APVQETLHGC QPVTQEDGKE SRISVQERQ SEQ ID NO: 42vector clone: pIB1010 Sequence:MALPVTALLL PLALLLHAAR PEQKLISEED LEIVLTQSPA TLSLSPGERATLSCRASKGV STSGYSYLHW YQQKPGQAPR LLIYLASYLE SGVPARFSGSGSGTDFTLTI SSLEPEDFAV YYCQHSRDLP LTFGGGTKVE IKRGGGGSGGGGSGGGGSQV QLVQSGVEVK KPGASVKVSC KASGYTFTNY YMYWVRQAPGQGLEWMGGIN PSNGGTNFNE KFKNRVTLTT DSSTTTAYME LKSLQFDDTAVYYCARRDYR FDMGFDYWGQ GTTVTVSSAA AGSGGSGILV KQSPMLVAYDNAVNLSCKYS YNLFSREFRA SLHKGLDSAV EVCVVYGNYS QQLQVYSKTGFNCDGKLGNE SVTFYLQNLY VNQTDIYFCK IEVMYPPPYL DNEKSNGTIIHVKGKHLCPS PLFPGPSKPF WVLVVVGGVL ACYSLLVTVA FIIFWVRSKRSRLLHSDYMN MTPRRPGPTR KHYQPYAPPR DFAAYRSKKV AKKPTNKAPHPKQEPQEINF PDDLPGSNTA APVQETLHGC QPVTQEDGKE SRISVQERQ SEQ ID NO: 43vector clone: pIB1011 Sequence:MALPVTALLL PLALLLHAAR PEQKLISEED LEVQLVESGG GLVQPGGSLRLSCAASGFTF SDSWIHWVRQ APGKGLEWVA WISPYGGSTY YADSVKGRFTISADTSKNTA YLQMNSLRAE DTAVYYCARR HWPGGFDYWG QGTLVTVSSGGGGSGGGGSG GGGSDIQMTQ SPSSLSASVG DRVTITCRAS QDVSTAVAWYQQKPGKAPKL LIYSASFLYS GVPSRFSGSG SGTDFTLTIS SLQPEDFATYYCQQYLYHPA TFGQGTKVEI KRAAAGSGGS GILVKQSPML VAYDNAVNLSCKYSYNLFSR EFRASLHKGL DSAVEVCVVY GNYSQQLQVY SKTGFNCDGKLGNESVTFYL QNLYVNQTDI YFCKIEVMYP PPYLDNEKSN GTIIHVKGKHLCPSPLFPGP SKPFWVLVVV GGVLACYSLL VTVAFIIFWV RSKRSRLLHSDYMNMTPRRP GPTRKHYQPY APPRDFAAYR SKKVAKKPTN KAPHPKQEPQEINFPDDLPG SNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ SEQ ID NO: 44vector clone: pIB1012 Sequence:MALPVTALLL PLALLLHAAR PEQKLISEED LDIQMTQSPS SLSASVGDRVTITCRASQDV STAVAWYQQK PGKAPKLLIY SASFLYSGVP SRFSGSGSGTDFTLTISSLQ PEDFATYYCQ QYLYHPATFG QGTKVEIKRG GGGSGGGGSGGGGSEVQLVE SGGGLVQPGG SLRLSCAASG FTFSDSWIHW VRQAPGKGLEWVAWISPYGG STYYADSVKG RFTISADTSK NTAYLQMNSL RAEDTAVYYCARRHWPGGFD YWGQGTLVTV SSAAAGSGGS GILVKQSPML VAYDNAVNLSCKYSYNLFSR EFRASLHKGL DSAVEVCVVY GNYSQQLQVY SKTGFNCDGKLGNESVTFYL QNLYVNQTDI YFCKIEVMYP PPYLDNEKSN GTIIHVKGKHLCPSPLFPGP SKPFWVLVVV GGVLACYSLL VTVAFIIFWV RSKRSRLLHSDYMNMTPRRP GPTRKHYQPY APPRDFAAYR SKKVAKKPTN KAPHPKQEPQEINFPDDLPG SNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ SEQ ID NO: 45vector clone: pIB1013 Sequence:MALPVTALLL PLALLLHAAR PEQKLISEED LQVQLVQSGA EVKKPGASVKVSCKASGYTF TNYWIGWVKQ APGQGLEWIG YLYPGGLYTN YNEKFKGKATMTADTSTNTA YMELSSLRSE DTAVYYCARY RDYDYAMDYW GQGTLVTVSSGGGGSGGGGS GGGGSDVVMT QTPLSLPVTL GQPASISCKS TKSLLNSDGFTYLGWCLQKP GQSPQLLIYL VSNRFSGVPD RFSGSGSGTD FTLKISRVEAEDVGVYYCFQ SNYLPLTFGQ GTKLEIKRAA AGSGGSGILV KQSPMLVAYDNAVNLSCKYS YNLFSREFRA SLHKGLDSAV EVCVVYGNYS QQLQVYSKTGFNCDGKLGNE SVTFYLQNLY VNQTDIYFCK IEVMYPPPYL DNEKSNGTIIHVKGKHLCPS PLFPGPSKPF WVLVVVGGVL ACYSLLVTVA FIIFWVRSKRSRLLHSDYMN MTPRRPGPTR KHYQPYAPPR DFAAYRSKKV AKKPTNKAPHPKQEPQEINF PDDLPGSNTA APVQETLHGC QPVTQEDGKE SRISVQERQ SEQ ID NO: 46vector clone: pIB1014 Sequence:MALPVTALLL PLALLLHAAR PEQKLISEED LDVVMTQTPL SLPVTLGQPASISCKSTKSL LNSDGFTYLG WCLQKPGQSP QLLIYLVSNR FSGVPDRFSGSGSGTDFTLK ISRVEAEDVG VYYCFQSNYL PLTFGQGTKL EIKRGGGGSGGGGSGGGGSQ VQLVQSGAEV KKPGASVKVS CKASGYTFTN YWIGWVKQAPGQGLEWIGYL YPGGLYTNYN EKFKGKATMT ADTSTNTAYM ELSSLRSEDTAVYYCARYRD YDYAMDYWGQ GTLVTVSSAA AGSGGSGILV KQSPMLVAYDNAVNLSCKYS YNLFSREFRA SLHKGLDSAV EVCVVYGNYS QQLQVYSKTGFNCDGKLGNE SVTFYLQNLY VNQTDIYFCK IEVMYPPPYL DNEKSNGTIIHVKGKHLCPS PLFPGPSKPF WVLVVVGGVL ACYSLLVTVA FIIFWVRSKRSRLLHSDYMN MTPRRPGPTR KHYQPYAPPR DFAAYRSKKV AKKPTNKAPHPKQEPQEINF PDDLPGSNTA APVQETLHGC QPVTQEDGKE SRISVQERQ SEQ ID NO: 47vector clone: pIB1015 Sequence:MALPVTALLL PLALLLHAAR PEQKLISEED LQVQLQESGP GLVKPSQTLSLTCAVYGGSF SSGYWNWIRK HPGKGLEYIG YISYNGITYH NPSLKSRITINRDTSKNQYS LQLNSVTPED TAVYYCARYK YDYDGGHAMD YWGQGTLVTVSSGGGGSGGG GSGGGGSDIQ MTQSPSSLSA SVGDRVTITC RASQDISNYLNWYQQKPGKA PKLLIYYTSK LHSGVPSRFS GSGSGTDYTL TISSLQPEDFATYYCQQGSA LPWTFGQGTK VEIKAAAGSG GSGILVKQSP MLVAYDNAVNLSCKYSYNLF SREFRASLHK GLDSAVEVCV VYGNYSQQLQ VYSKTGFNCDGKLGNESVTF YLQNLYVNQT DIYFCKIEVM YPPPYLDNEK SNGTIIHVKGKHLCPSPLFP GPSKPFWVLV VVGGVLACYS LLVTVAFIIF WVRSKRSRLLHSDYMNMTPR RPGPTRKHYQ PYAPPRDFAA YRSKKVAKKP TNKAPHPKQEPQEINFPDDL PGSNTAAPVQ ETLHGCQPVT QEDGKESRIS VQERQ SEQ ID NO: 48vector clone: pIB1016 Sequence:MALPVTALLL PLALLLHAAR PEQKLISEED LDIQMTQSPS SLSASVGDRVTITCRASQDI SNYLNWYQQK PGKAPKLLIY YTSKLHSGVP SRFSGSGSGTDYTLTISSLQ PEDFATYYCQ QGSALPWTFG QGTKVEIKGG GGSGGGGSGGGGSQVQLQES GPGLVKPSQT LSLTCAVYGG SFSSGYWNWI RKHPGKGLEYIGYISYNGIT YHNPSLKSRI TINRDTSKNQ YSLQLNSVTP EDTAVYYCARYKYDYDGGHA MDYWGQGTLV TVSSAAAGSG GSGILVKQSP MLVAYDNAVNLSCKYSYNLF SREFRASLHK GLDSAVEVCV VYGNYSQQLQ VYSKTGFNCDGKLGNESVTF YLQNLYVNQT DIYFCKIEVM YPPPYLDNEK SNGTIIHVKGKHLCPSPLFP GPSKPFWVLV VVGGVLACYS LLVTVAFIIF WVRSKRSRLLHSDYMNMTPR RPGPTRKHYQ PYAPPRDFAA YRSKKVAKKP TNKAPHPKQEPQEINFPDDL PGSNTAAPVQ ETLHGCQPVT QEDGKESRIS VQERQ SEQ ID NO: 49vector clone: pIB1017 Sequence:MALPVTALLL PLALLLHAAR PEQKLISEED LQVQLVESGG GVVQPGRSLRLSCAASGFTF SSYTMHWVRQ APGKGLEWVT FISYDGNNKY YADSVKGRFTISRDNSKNTL YLQMNSLRAE DTAIYYCART GWLGPFDYWG QGTLVTVSSGGGGSGGGGSG GGGSEIVLTQ SPGTLSLSPG ERATLSCRAS QSVGSSYLAWYQQKPGQAPR LLIYGAFSRA TGIPDRFSGS GSGTDFTLTI SRLEPEDFAVYYCQQYGSSP WTFGQGTKVE IKAAAGSGGS GILVKQSPML VAYDNAVNLSCKYSYNLFSR EFRASLHKGL DSAVEVCVVY GNYSQQLQVY SKTGFNCDGKLGNESVTFYL QNLYVNQTDI YFCKIEVMYP PPYLDNEKSN GTIIHVKGKHLCPSPLFPGP SKPFWVLVVV GGVLACYSLL VTVAFIIFWV RSKRSRLLHSDYMNMTPRRP GPTRKHYQPY APPRDFAAYR SKKVAKKPTN KAPHPKQEPQEINFPDDLPG SNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ SEQ ID NO: 50vector clone: pIB1018 Sequence:MALPVTALLL PLALLLHAAR PEQKLISEED LEIVLTQSPG TLSLSPGERATLSCRASQSV GSSYLAWYQQ KPGQAPRLLI YGAFSRATGI PDRFSGSGSGTDFTLTISRL EPEDFAVYYC QQYGSSPWTF GQGTKVEIKG GGGSGGGGSGGGGSQVQLVE SGGGVVQPGR SLRLSCAASG FTFSSYTMHW VRQAPGKGLEWVTFISYDGN NKYYADSVKG RFTISRDNSK NTLYLQMNSL RAEDTAIYYCARTGWLGPFD YWGQGTLVTV SSAAAGSGGS GILVKQSPML VAYDNAVNLSCKYSYNLFSR EFRASLHKGL DSAVEVCVVY GNYSQQLQVY SKTGFNCDGKLGNESVTFYL QNLYVNQTDI YFCKIEVMYP PPYLDNEKSN GTIIHVKGKHLCPSPLFPGP SKPFWVLVVV GGVLACYSLL VTVAFIIFWV RSKRSRLLHSDYMNMTPRRP GPTRKHYQPY APPRDFAAYR SKKVAKKPTN KAPHPKQEPQEINFPDDLPG SNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ SEQ ID NO: 51vector clone: pIB1019 Sequence:MALPVTALLL PLALLLHAAR PEQKLISEED LEVQLVESGG GVVRPGGSLRLSCVASGVTF DDYGMSWVRQ APGKGLEWVS GINWNGGDTD YSDSVKGRFTISRDNAKNSL YLQMNSLRAE DTALYYCARD FYGSGSYYHV PFDYWGQGILVTVSSGGGGS GGGGSGGGGS EIVLTQSPGT LSLSPGERAT LSCRASQSVSRSYLAWYQQK RGQAPRLLIY GASSRATGIP DRFSGDGSGT DFTLSISRLEPEDFAVYYCH QYDMSPFTFG PGTKVDIKAA AGSGGSGILV KQSPMLVAYDNAVNLSCKYS YNLFSREFRA SLHKGLDSAV EVCVVYGNYS QQLQVYSKTGFNCDGKLGNE SVTFYLQNLY VNQTDIYFCK IEVMYPPPYL DNEKSNGTIIHVKGKHLCPS PLFPGPSKPF WVLVVVGGVL ACYSLLVTVA FIIFWVRSKRSRLLHSDYMN MTPRRPGPTR KHYQPYAPPR DFAAYRSKKV AKKPTNKAPHPKQEPQEINF PDDLPGSNTA APVQETLHGC QPVTQEDGKE SRISVQERQ SEQ ID NO: 52vector clone: pIB1020 Sequence:MALPVTALLL PLALLLHAAR PEQKLISEED LEIVLTQSPG TLSLSPGERATLSCRASQSV SRSYLAWYQQ KRGQAPRLLI YGASSRATGI PDRFSGDGSGTDFTLSISRL EPEDFAVYYC HQYDMSPFTF GPGTKVDIKG GGGSGGGGSGGGGSEVQLVE SGGGVVRPGG SLRLSCVASG VTFDDYGMSW VRQAPGKGLEWVSGINWNGG DTDYSDSVKG RFTISRDNAK NSLYLQMNSL RAEDTALYYCARDFYGSGSY YHVPFDYWGQ GILVTVSSAA AGSGGSGILV KQSPMLVAYDNAVNLSCKYS YNLFSREFRA SLHKGLDSAV EVCVVYGNYS QQLQVYSKTGFNCDGKLGNE SVTFYLQNLY VNQTDIYFCK IEVMYPPPYL DNEKSNGTIIHVKGKHLCPS PLFPGPSKPF WVLVVVGGVL ACYSLLVTVA FIIFWVRSKRSRLLHSDYMN MTPRRPGPTR KHYQPYAPPR DFAAYRSKKV AKKPTNKAPHPKQEPQEINF PDDLPGSNTA APVQETLHGC QPVTQEDGKE SRISVQERQ SEQ ID NO: 53vector clone: pIB1021 Sequence:MALPVTALLL PLALLLHAAR PEQKLISEED LQVQLVESGG GVVQPGRSLRLSCAASGFSF SSTYVCWVRQ APGKGLEWIA CIYTGDGTNY SASWAKGRFTISKDSSKNTV YLQMNSLRAE DTAVYFCARP DITYGFAINF WGPGTLVTVSSGGGGSGGGG SGGGGSDIQM TQSPSSLSAS VGDRVTIKCQ ASQSISSRLAWYQQKPGKPP KLLIYRASTL ASGVPSRFSG SGSGTDFTLT ISSLQPEDVATYYCQCTGYG ISWPIGGGTK VEIKAAAGSG GSGILVKQSP MLVAYDNAVNLSCKYSYNLF SREFRASLHK GLDSAVEVCV VYGNYSQQLQ VYSKTGFNCDGKLGNESVTF YLQNLYVNQT DIYFCKIEVM YPPPYLDNEK SNGTIIHVKGKHLCPSPLFP GPSKPFWVLV VVGGVLACYS LLVTVAFIIF WVRSKRSRLLHSDYMNMTPR RPGPTRKHYQ PYAPPRDFAA YRSKKVAKKP TNKAPHPKQEPQEINFPDDL PGSNTAAPVQ ETLHGCQPVT QEDGKESRIS VQERQ SEQ ID NO: 54vector clone: pIB1022 Sequence:MALPVTALLL PLALLLHAAR PEQKLISEED LDIQMTQSPS SLSASVGDRVTIKCQASQSI SSRLAWYQQK PGKPPKLLIY RASTLASGVP SRFSGSGSGTDFTLTISSLQ PEDVATYYCQ CTGYGISWPI GGGTKVEIKG GGGSGGGGSGGGGSQVQLVE SGGGVVQPGR SLRLSCAASG FSFSSTYVCW VRQAPGKGLEWIACIYTGDG TNYSASWAKG RFTISKDSSK NTVYLQMNSL RAEDTAVYFCARPDITYGFA INFWGPGTLV TVSSAAAGSG GSGILVKQSP MLVAYDNAVNLSCKYSYNLF SREFRASLHK GLDSAVEVCV VYGNYSQQLQ VYSKTGFNCDGKLGNESVTF YLQNLYVNQT DIYFCKIEVM YPPPYLDNEK SNGTIIHVKGKHLCPSPLFP GPSKPFWVLV VVGGVLACYS LLVTVAFIIF WVRSKRSRLLHSDYMNMTPR RPGPTRKHYQ PYAPPRDFAA YRSKKVAKKP TNKAPHPKQEPQEINFPDDL PGSNTAAPVQ ETLHGCQPVT QEDGKESRIS VQERQ SEQ ID NO: 55vector clone: pIB1023 Sequence:MALPVTALLL PLALLLHAAR PEQKLISEED LQVQLVQSGA EVKKPGASVKVSCKASGYTF TGYYMHWVRQ APGQGLEWMG WINPDSGGTN YAQKFQGRVTMTRDTSISTA YMELNRLRSD DTAVYYCARD QPLGYCTNGV CSYFDYWGQGTLVTVSSGGG GSGGGGSGGG GSDIQMTQSP SSVSASVGDR VTITCRASQGIYSWLAWYQQ KPGKAPNLLI YTASTLQSGV PSRFSGSGSG TDFTLTISSLQPEDFATYYC QQANIFPLTF GGGTKVEIKA AAGSGGSGIL VKQSPMLVAYDNAVNLSCKY SYNLFSREFR ASLHKGLDSA VEVCVVYGNY SQQLQVYSKTGFNCDGKLGN ESVTFYLQNL YVNQTDIYFC KIEVMYPPPY LDNEKSNGTIIHVKGKHLCP SPLFPGPSKP FWVLVVVGGV LACYSLLVTV AFIIFWVRSKRSRLLHSDYM NMTPRRPGPT RKHYQPYAPP RDFAAYRSKK VAKKPTNKAPHPKQEPQEIN FPDDLPGSNT AAPVQETLHG CQPVTQEDGK ESRISVQERQ SEQ ID NO: 56vector clone: pIB1024 Sequence:MALPVTALLL PLALLLHAAR PEQKLISEED LDIQMTQSPS SVSASVGDRVTITCRASQGI YSWLAWYQQK PGKAPNLLIY TASTLQSGVP SRFSGSGSGTDFTLTISSLQ PEDFATYYCQ QANIFPLTFG GGTKVEIKGG GGSGGGGSGGGGSQVQLVQS GAEVKKPGAS VKVSCKASGY TFTGYYMHWV RQAPGQGLEWMGWINPDSGG TNYAQKFQGR VTMTRDTSIS TAYMELNRLR SDDTAVYYCARDQPLGYCTN GVCSYFDYWG QGTLVTVSSA AAGSGGSGIL VKQSPMLVAYDNAVNLSCKY SYNLFSREFR ASLHKGLDSA VEVCVVYGNY SQQLQVYSKTGFNCDGKLGN ESVTFYLQNL YVNQTDIYFC KIEVMYPPPY LDNEKSNGTIIHVKGKHLCP SPLFPGPSKP FWVLVVVGGV LACYSLLVTV AFIIFWVRSKRSRLLHSDYM NMTPRRPGPT RKHYQPYAPP RDFAAYRSKK VAKKPTNKAPHPKQEPQEIN FPDDLPGSNT AAPVQETLHG CQPVTQEDGK ESRISVQERQ SEQ ID NO: 57vector clone: pIB1025 Sequence:MALPVTALLL PLALLLHAAR PEQKLISEED LILVKQSPML VAYDNAVNLSCKYSYNLFSR EFRASLHKGL DSAVEVCVVY GNYSQQLQVY SKTGFNCDGKLGNESVTFYL QNLYVNQTDI YFCKIEVMYP PPYLDNEKSN GTIIHVKGKHLCPSPLFPGP SKPFWVLVVV GGVLACYSLL VTVAFIIFWV RSKRSRLLHSDYMNMTPRRP GPTRKHYQPY APPRDFAAYR SKKVAKKPTN KAPHPKQEPQEINFPDDLPG SNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ SEQ ID NO: 58vector clone: pIB1026 Sequence:MEHSTFLSGL VLATLLSQVS PEQKLISEED LFKIPIEELE DRVFVNCNTSITWVEGTVGT LLSDITRLDL GKRILDPRGI YRCNGTDIYK DKESTVQVHYRMCQSCVELD PATVAGIIVT DVIATLLLAL GVFCFARSKR SRLLHSDYMNMTPRRPGPTR KHYQPYAPPR DFAAYRSKKV AKKPTNKAPH PKQEPQEINFPDDLPGSNTA APVQETLHGC QPVTQEDGKE SRISVQERQ SEQ ID NO: 59 vector clone:pIB1027 Sequence: MQSGTHWRVL GLCLLSVGVW GQDYKDDDDK DGNEEMGGIT QTPYKVSISGTTVILTCPQY PGSEILWQHN DKNIGGDEDD KNIGSDEDHL SLKEFSELEQSGYYVCYPRG SKPEDANFYL YLRARVCENC MEMDVMSVAT IVIVDICITGGLLLLVYYWS RSKRSRLLHS DYMNMTPRRP GPTRKHYQPY APPRDFAAYRSKKVAKKPTN KAPHPKQEPQ EINFPDDLPG SNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQSEQ ID NO: 60 vector clone: pIB1028 Sequence:MEQGKGLAVL ILAIILLQGT LAEQKLISEE DLQSIKGNHL VKVYDYQEDGSVLLTCDAEA KNITWFKDGK MIGFLTEDKK KWNLGSNAKD PRGMYQCKGSQNKSKPLQVY YRMCQNCIEL NAATISGFLF AEIVSIFVLA VGVYFIARSKRSRLLHSDYM NMTPRRPGPT RKHYQPYAPP RDFAAYRSKK VAKKPTNKAPHPKQEPQEIN FPDDLPGSNT AAPVQETLHG CQPVTQEDGK ESRISVQERQ SEQ ID NO: 61vector clone: pIB1029 Sequence:MKWKALFTAA ILQAQLPITE AQSFGLLDPK LCYLLDGILF IYGVILTALFLRSKRSRLLH SDYMNMTPRR PGPTRKHYQP YAPPRDFAAY RSKKVAKKPTNKAPHPKQEP QEINFPDDLP GSNTAAPVQE TLHGCQPVTQ EDGKESRISV QERQEQKLIS EEDLSEQ ID NO: 62 vector clone: pIB1030 Sequence:MALPVTALLL PLALLLHAAR PEQKLISEED LSQPHTKPSV FVMKNGTNVACLVKEFYPKD IRINLVSSKK ITEFDPAIVI SPSGKYNAVK LGKYEDSNSVTCSVQHDNKT VHSTDFEVKT DSTDHVKPKE TENTKQPSKS CHKPKAIVHTEKVNMMSLTV LGLRMLFAKT VAVNFLLTAK LFFLRSKRSR LLHSDYMNMTPRRPGPTRKH YQPYAPPRDF AAYRSKKVAK KPTNKAPHPK QEPQEINFPDDLPGSNTAAP VQETLHGCQP VTQEDGKESR ISVQERQ SEQ ID NO: 63 vector clone:pIB1031 Sequence: MALPVTALLL PLALLLHAAR PDYKDDDDKD KQLDADVSPK PTIFLPSIAETKLQKAGTYL CLLEKFFPDV IKIHWQEKKS NTILGSQEGN TMKTNDTYMKFSWLTVPEKS LDKEHRCIVR HENNKNGVDQ EIIFPPIKTD VITMDPKDNCSKDANDTLLL QLTNTSAYYM YLLLLLKSVV YFAIITCCLL RSKRSRLLHSDYMNMTPRRP GPTRKHYQPY APPRDFAAYR SKKVAKKPTN KAPHPKQEPQEINFPDDLPG SNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ SEQ ID NO: 64vector clone: pIB1032 Sequence:MALPVTALLL PLALLLHAAR PEQKLISEED LIQNPEPAVY QLKDPRSQDSTLCLFTDFDS QINVPKTMES GTFITDKCVL DMKAMDSKSN GAIAWSNQTSFTCQDIFKET NATYPSSDVP CDATLTEKSF ETDMNLNFQN LLVIVLRILLLKVAGFNLLM TLRLWSSRSK RSRLLHSDYM NMTPRRPGPT RKHYQPYAPPRDFAAYRSKK VAKKPTNKAP HPKQEPQEIN FPDDLPGSNT AAPVQETLHGCQPVTQEDGK ESRISVQERQ SEQ ID NO: 65 vector clone: pIB1033 Sequence:MALPVTALLL PLALLLHAAR PDYKDDDDKV LTPPKVSLFE PSKAEIANKQKATLVCLARG FFPDHVELSW WVNGKEVHSG VCTDPQAYKE SNYSYCLSSRLRVSATFWHN PRNHFRCQVQ FHGLSEEDKW PEGSPKPVTQ NISAEAWGRADCGITSASYQ QGVLSATILY EILLGKATLY AVLVSTLVVM AMVKRKNSRSKRSRLLHSDY MNMTPRRPGP TRKHYQPYAP PRDFAAYRSK KVAKKPTNKAPHPKQEPQEI NFPDDLPGSN TAAPVQETLH GCQPVTQEDG KESRISVQER Q SEQ ID NO: 66vector clone: pIB1046 Sequence:MALPVTALLL PLALLLHAAR PEQKLISEED LSQPHTKPSV FVMKNGTNVACLVKEFYPKD IRINLVSSKK ITEFDPAIVI SPSGKYNAVK LGKYEDSNSVTCSVQHDNKT VHSTDFEVKT DSTDHVKPKE TENTKQPSKS CHKPKAIVHTEKVNMMSLTV LGLRMLFAKT VAVNFLLTAK LFFLRSKRSR LLHSDYMNMTPRRPGPTRKH YQPYAPPRDF AAYRSKKVAK KPTNKAPHPK QEPQEINFPDDLPGSNTAAP VQETLHGCQP VTQEDGKESR ISVQERQRAK RGSGEGRGSLLTCGDVEENP GPMALPVTAL LLPLALLLHA ARPDYKDDDD KDKQLDADVSPKPTIFLPSI AETKLQKAGT YLCLLEKFFP DVIKIHWQEK KSNTILGSQEGNTMKTNDTY MKFSWLTVPE KSLDKEHRCI VRHENNKNGV DQEIIFPPIKTDVITMDPKD NCSKDANDTL LLQLTNTSAY YMYLLLLLKS VVYFAIITCCLLRSKRSRLL HSDYMNMTPR RPGPTRKHYQ PYAPPRDFAA YRSKKVAKKPTNKAPHPKQE PQEINFPDDL PGSNTAAPVQ ETLHGCQPVT QEDGKESRIS VQERQSEQ ID NO: 67 vector clone: pIB1047 Sequence:MALPVTALLL PLALLLHAAR PEQKLISEED LIQNPEPAVY QLKDPRSQDSTLCLFTDFDS QINVPKTMES GTFITDKCVL DMKAMDSKSN GAIAWSNQTSFTCQDIFKET NATYPSSDVP CDATLTEKSF ETDMNLNFQN LLVIVLRILLLKVAGFNLLM TLRLWSSRSK RSRLLHSDYM NMTPRRPGPT RKHYQPYAPPRDFAAYRSKK VAKKPTNKAP HPKQEPQEIN FPDDLPGSNT AAPVQETLHGCQPVTQEDGK ESRISVQERQ RAKRGSGEGR GSLLTCGDVE ENPGPMALPVTALLLPLALL LHAARPDYKD DDDKVLTPPK VSLFEPSKAE IANKQKATLVCLARGFFPDH VELSWWVNGK EVHSGVCTDP QAYKESNYSY CLSSRLRVSATFWHNPRNHF RCQVQFHGLS EEDKWPEGSP KPVTQNISAE AWGRADCGITSASYQQGVLS ATILYEILLG KATLYAVLVS TLVVMAMVKR KNSRSKRSRLLHSDYMNMTP RRPGPTRKHY QPYAPPRDFA AYRSKKVAKK PTNKAPHPKQEPQEINFPDD LPGSNTAAPV QETLHGCQPV TQEDGKESRI SVQERQ SEQ ID NO: 68vector clone: pIB1048 Sequence:MEHSTFLSGL VLATLLSQVS PEQKLISEED LFKIPIEELE DRVFVNCNTSITWVEGTVGT LLSDITRLDL GKRILDPRGI YRCNGTDIYK DKESTVQVHYRMCQSCVELD PATVAGIIVT DVIATLLLAL GVFCFARSKR SRLLHSDYMNMTPRRPGPTR KHYQPYAPPR DFAAYRSKKV AKKPTNKAPH PKQEPQEINFPDDLPGSNTA APVQETLHGC QPVTQEDGKE SRISVQERQR AKRGSGEGRGSLLTCGDVEE NPGPMQSGTH WRVLGLCLLS VGVWGQDYKD DDDKDGNEEMGGITQTPYKV SISGTTVILT CPQYPGSEIL WQHNDKNIGG DEDDKNIGSDEDHLSLKEFS ELEQSGYYVC YPRGSKPEDA NFYLYLRARV CENCMEMDVMSVATIVIVDI CITGGLLLLV YYWSRSKRSR LLHSDYMNMT PRRPGPTRKHYQPYAPPRDF AAYRSKKVAK KPTNKAPHPK QEPQEINFPD DLPGSNTAAPVQETLHGCQP VTQEDGKESR ISVQERQ SEQ ID NO: 69 vector clone: pIB1049Sequence: MEQGKGLAVL ILAIILLQGT LAEQKLISEE DLQSIKGNHL VKVYDYQEDGSVLLTCDAEA KNITWFKDGK MIGFLTEDKK KWNLGSNAKD PRGMYQCKGSQNKSKPLQVY YRMCQNCIEL NAATISGFLF AEIVSIFVLA VGVYFIARSKRSRLLHSDYM NMTPRRPGPT RKHYQPYAPP RDFAAYRSKK VAKKPTNKAPHPKQEPQEIN FPDDLPGSNT AAPVQETLHG CQPVTQEDGK ESRISVQERQRAKRGSGMQS GTHWRVLGLC LLSVGVWGQD YKDDDDKDGN EEMGGITQTPYKVSISGTTV ILTCPQYPGS EILWQHNDKN IGGDEDDKNI GSDEDHLSLKEFSELEQSGY YVCYPRGSKP EDANFYLYLR ARVCENCMEM DVMSVATIVIVDICITGGLL LLVYYWSRSK RSRLLHSDYM NMTPRRPGPT RKHYQPYAPPRDFAAYRSKK VAKKPTNKAP HPKQEPQEIN FPDDLPGSNT AAPVQETLHGCQPVTQEDGK ESRISVQERQ SEQ ID NO: 70 vector clone: pIB1050 Sequence:MEHSTFLSGL VLATLLSQVS PEQKLISEED LFKIPIEELE DRVFVNCNTSITWVEGTVGT LLSDITRLDL GKRILDPRGI YRCNGTDIYK DKESTVQVHYRMCQSCVELD PATVAGIIVT DVIATLLLAL GVFCFAGHET GRLSGAADTQALLRNDQVYQ PLRDRDDAQY SHLGGNWARN KRSKRSRLLH SDYMNMTPRRPGPTRKHYQP YAPPRDFAAY RSKKVAKKPT NKAPHPKQEP QEINFPDDLPGSNTAAPVQE TLHGCQPVTQ EDGKESRISV QERQRAKRGS GEGRGSLLTCGDVEENPGPM QSGTHWRVLG LCLLSVGVWG QDYKDDDDKD GNEEMGGITQTPYKVSISGT TVILTCPQYP GSEILWQHND KNIGGDEDDK NIGSDEDHLSLKEFSELEQS GYYVCYPRGS KPEDANFYLY LRARVCENCM EMDVMSVATIVIVDICITGG LLLLVYYWSK NRKAKAKPVT RGAGAGGRQR GQNKERPPPVPNPDYEPIRK GQRDLYSGLN QRRIRSKRSR LLHSDYMNMT PRRPGPTRKHYQPYAPPRDF AAYRSKKVAK KPTNKAPHPK QEPQEINFPD DLPGSNTAAPVQETLHGCQP VTQEDGKESR ISVQERQ SEQ ID NO: 71 vector clone: pIB1051Sequence: MEHSTFLSGL VLATLLSQVS PEQKLISEED LFKIPIEELE DRVFVNCNTSITWVEGTVGT LLSDITRLDL GKRILDPRGI YRCNGTDIYK DKESTVQVHYRMCQSCVELD PATVAGIIVT DVIATLLLAL GVFCFAGHET GRLSGAADTQALLRNDQVYQ PLRDRDDAQY SHLGGNWARN KRAKRGSGEG RGSLLTCGDVEENPGPMQSG THWRVLGLCL LSVGVWGQDY KDDDDKDGNE EMGGITQTPYKVSISGTTVI LTCPQYPGSE ILWQHNDKNI GGDEDDKNIG SDEDHLSLKEFSELEQSGYY VCYPRGSKPE DANFYLYLRA RVCENCMEMD VMSVATIVIVDICITGGLLL LVYYWSKNRK AKAKPVTRGA GAGGRQRGQN KERPPPVPNPDYEPIRKGQR DLYSGLNQRR I SEQ ID NO: 72 vector clone: pIB1052 Sequence:MEHSTFLSGL VLATLLSQVS PEQKLISEED LFKIPIEELE DRVFVNCNTSITWVEGTVGT LLSDITRLDL GKRILDPRGI YRCNGTDIYK DKESTVQVHYRMCQSCVELD PATVAGIIVT DVIATLLLAL GVFCFARAKR GSGEGRGSLLTCGDVEENPG PMQSGTHWRV LGLCLLSVGV WGQDYKDDDD KDGNEEMGGITQTPYKVSIS GTTVILTCPQ YPGSEILWQH NDKNIGGDED DKNIGSDEDHLSLKEFSELE QSGYYVCYPR GSKPEDANFY LYLRARVCEN CMEMDVMSVATIVIVDICIT GGLLLLVYYW S SEQ ID NO: 73 vector clone: pIB1053 Sequence:MEQGKGLAVL ILAIILLQGT LAEQKLISEE DLQSIKGNHL VKVYDYQEDGSVLLTCDAEA KNITWFKDGK MIGFLTEDKK KWNLGSNAKD PRGMYQCKGSQNKSKPLQVY YRMCQNCIEL NAATISGFLF AEIVSIFVLA VGVYFIAGQDGVRQSRASDK QTLLPNDQLY QPLKDREDDQ YSHLQGNQLR RNRSKRSRLLHSDYMNMTPR RPGPTRKHYQ PYAPPRDFAA YRSKKVAKKP TNKAPHPKQEPQEINFPDDL PGSNTAAPVQ ETLHGCQPVT QEDGKESRIS VQERQRAKRGSGEGRGSLLT CGDVEENPGP MQSGTHWRVL GLCLLSVGVW GQDYKDDDDKDGNEEMGGIT QTPYKVSISG TTVILTCPQY PGSEILWQHN DKNIGGDEDDKNIGSDEDHL SLKEFSELEQ SGYYVCYPRG SKPEDANFYL YLRARVCENCMEMDVMSVAT IVIVDICITG GLLLLVYYWS KNRKAKAKPV TRGAGAGGRQRGQNKERPPP VPNPDYEPIR KGQRDLYSGL NQRRIRSKRS RLLHSDYMNMTPRRPGPTRK HYQPYAPPRD FAAYRSKKVA KKPTNKAPHP KQEPQEINFPDDLPGSNTAA PVQETLHGCQ PVTQEDGKES RISVQERQ SEQ ID NO: 74 vector clone:pIB1054 Sequence: MEQGKGLAVL ILAIILLQGT LAEQKLISEE DLQSIKGNHL VKVYDYQEDGSVLLTCDAEA KNITWFKDGK MIGFLTEDKK KWNLGSNAKD PRGMYQCKGSQNKSKPLQVY YRMCQNCIEL NAATISGFLF AEIVSIFVLA VGVYFIAGQDGVRQSRASDK QTLLPNDQLY QPLKDREDDQ YSHLQGNQLR RNRAKRGSGEGRGSLLTCGD VEENPGPMQS GTHWRVLGLC LLSVGVWGQD YKDDDDKDGNEEMGGITQTP YKVSISGTTV ILTCPQYPGS EILWQHNDKN IGGDEDDKNIGSDEDHLSLK EFSELEQSGY YVCYPRGSKP EDANFYLYLR ARVCENCMEMDVMSVATIVI VDICITGGLL LLVYYWSKNR KAKAKPVTRG AGAGGRQRGQNKERPPPVPN PDYEPIRKGQ RDLYSGLNQR RI SEQ ID NO: 75 vector clone: pIB1055Sequence: MEQGKGLAVL ILAIILLQGT LAEQKLISEE DLQSIKGNHL VKVYDYQEDGSVLLTCDAEA KNITWFKDGK MIGFLTEDKK KWNLGSNAKD PRGMYQCKGSQNKSKPLQVY YRMCQNCIEL NAATISGFLF AEIVSIFVLA VGVYFIARAKRGSGEGRGSL LTCGDVEENP GPMQSGTHWR VLGLCLLSVG VWGQDYKDDDDKDGNEEMGG ITQTPYKVSI SGTTVILTCP QYPGSEILWQ HNDKNIGGDEDDKNIGSDED HLSLKEFSEL EQSGYYVCYP RGSKPEDANF YLYLRARVCENCMEMDVMSV ATIVIVDICI TGGLLLLVYY WS SEQ ID NO: 76 vector clone: pIB1056Sequence: MKWKALFTAA ILQAQLPITE AQSFGLLDPK LCYLLDGILF IYGVILTALFLRVKFSRSAD APAYQQGQNQ LYNELNLGRR EEYDVLDKRR GRDPEMGGKPQRRKNPQEGL YNELQKDKMA EAYSEIGMKG ERRRGKGHDG LYQGLSTATKDTYDALHMQA LPPRRSKRSR LLHSDYMNMT PRRPGPTRKH YQPYAPPRDFAAYRSKKVAK KPTNKAPHPK QEPQEINFPD DLPGSNTAAP VQETLHGCQPVTQEDGKESR ISVQERQEQK LISEEDL SEQ ID NO: 78 vector clone: pIB1057Sequence: MKWKALFTAA ILQAQLPITE AQSFGLLDPK LCYLLDGILF IYGVILTALFLRVKFSRSAD APAYQQGQNQ LYNELNLGRR EEYDVLDKRR GRDPEMGGKPQRRKNPQEGL YNELQKDKMA EAYSEIGMKG ERRRGKGHDG LYQGLSTATKDTYDALHMQA LPPREQKLIS EEDL SEQ ID NO: 79 vector clone: pIB1058 Sequence:MKWKALFTAA ILQAQLPITE AQSFGLLDPK LCYLLDGILF IYGVILTALF LEQKLISEED LSEQ ID NO: 80 vector clone: pIB1059 Sequence:MKWKALFTAA ILQAQLPITE AQSFGLLDPK LCYLLDGILF IYGVILTALFLRVKFSRSAD APAYQQGQNQ LYNELNLGRR EEYDVLDKRR GRDPEMGGKPQRRKNPQEGL YNELQKDKMA EAYSEIGMKG ERRRGKGHDG LYQGLSTATKDTYDALHMQA LPPRRVKFSR SADAPAYQQG QNQLYNELNL GRREEYDVLDKRRGRDPEMG GKPQRRKNPQ EGLYNELQKD KMAEAYSEIG MKGERRRGKGHDGLYQGLST ATKDTYDALH MQALPPRRSK RSRLLHSDYM NMTPRRPGPTRKHYQPYAPP RDFAAYRSKK VAKKPTNKAP HPKQEPQEIN FPDDLPGSNTAAPVQETLHG CQPVTQEDGK ESRISVQERQ EQKLISEEDL SEQ ID NO: 81 vector clone:pIB1060 Sequence: MKWKALFTAA ILQAQLPITE AQSFGLLDPK LCYLLDGILF IYGVILTALFLRVKFSRSAD APAYQQGQNQ LYNELNLGRR EEYDVLDKRR GRDPEMGGKPQRRKNPQEGL YNELQKDKMA EAYSEIGMKG ERRRGKGHDG LYQGLSTATKDTYDALHMQA LPPRRVKFSR SADAPAYQQG QNQLYNELNL GRREEYDVLDKRRGRDPEMG GKPQRRKNPQ EGLYNELQKD KMAEAYSEIG MKGERRRGKGHDGLYQGLST ATKDTYDALH MQALPPREQK LISEEDL SEQ ID NO: 82 vector clone:pIB1061 Sequence: MKWKALFTAA ILQAQLPITE AQSFGLLDPK LCYLLDGILF IYGVILTALFLRSKRSRLLH SDYMNMTPRR PGPTRKHYQP YAPPRDFAAY RSKKVAKKPTNKAPHPKQEP QEINFPDDLP GSNTAAPVQE TLHGCQPVTQ EDGKESRISVQERQRVKFSR SADAPAYQQG QNQLYNELNL GRREEYDVLD KRRGRDPEMGGKPQRRKNPQ EGLYNELQKD KMAEAYSEIG MKGERRRGKG HDGLYQGLSTATKDTYDALH MQALPPRRVK FSRSADAPAY QQGQNQLYNE LNLGRREEYDVLDKRRGRDP EMGGKPQRRK NPQEGLYNEL QKDKMAEAYS EIGMKGERRRGKGHDGLYQG LSTATKDTYD ALHMQALPPR EQKLISEEDL SEQ ID NO: 83 vector clone:pIB1062 Sequence: MKWKALFTAA ILQAQLPITE AQSFGLLDPK LCYLLDGILF IYGVILTALFLRSKRSRLLH SDYMNMTPRR PGPTRKHYQP YAPPRDFAAY RSKKVAKKPTNKAPHPKQEP QEINFPDDLP GSNTAAPVQE TLHGCQPVTQ EDGKESRISVQERQRVKFSR SADAPAYQQG QNQLYNELNL GRREEYDVLD KRRGRDPEMGGKPQRRKNPQ EGLYNELQKD KMAEAYSEIG MKGERRRGKG HDGLYQGLSTATKDTYDALH MQALPPREQK LISEEDL SEQ ID NO: 84 vector clone: pIB1063Sequence: MGHTRRQGTS PSKCPYLNFF QLLVLAGLSH FCSGEQKLIS EEDLVIHVTKEVKEVATLSC GHNVSVEELA QTRIYWQKEK KMVLTMMSGD MNIWPEYKNRTIFDITNNLS IVILALRPSD EGTYECVVLK YEKDAFKREH LAEVTLSVKADFPTPSISDF EIPTSNIRRI ICSTSGGFPE PHLSWLENGE ELNAINTTVSQDPETELYAV SSKLDFNMTT NHSFMCLIKY GHLRVNQTFN WNTTKQEHFPDNLLPSWAIT LISVNGIFVI CCL SEQ ID NO: 85 vector clone: pIB1064 Sequence:MGCGCSSHPE DDWMENIDVC ENCHYPIVPL DGKGTLLIRN GSEVRDPLVTYEGSNPPASP LQDNLVIALH SYEPSHDGDL GFEKGEQLRI LEQSGEWWKAQSLTTGQEGF IPFNFVAKAN SLEPEPWFFK NLSRKDAERQ LLAPGNTHGSFLIRESESTA GSFSLSVRDF DQNQGEVVKH YKIRNLDNGG FYISPRITFPGLHELVRHYT NASDGLCTRL SRPCQTQKPQ KPWWEDEWEV PRETLKLVERLGAGQFGEVW MGYYNGHTKV AVKSLKQGSM SPDAFLAEAN LMKQLQHQRLVRLYAVVTQE PIYIITEYME NGSLVDFLKT PSGIKLTINK LLDMAAQIAEGMAFIEERNY IHRDLRAANI LVSDTLSCKI ADFGLARLIE DNEYTAREGAKFPIKWTAPE AINYGTFTIK SDVWSFGILL TEIVTHGRIP YPGMTNPEVIQNLERGYRMV RPDNCPEELY QLMRLCWKER PEDRPTFDYL RSVLEDFFTATEGQYQPQPE QKLISEEDL SEQ ID NO: 86 vector clone: pIB1065 Sequence:MGCGCSSHPE DDWMENIDVC ENCHYPIVPL DGKGTLLIRN GSEVRDPLVTYEGSNPPASP LQDNLVIALH SYEPSHDGDL GFEKGEQLRI LEQSGEWWKAQSLTTGQEGF IPFNFVAKAN SLEPEPWFFK NLSRKDAERQ LLAPGNTHGSFLIRESESTA GSFSLSVRDF DQNQGEVVKH YKIRNLDNGG FYISPRITFPGLHELVRHYT NASDGLCTRL SRPCQTQKPQ KPWWEDEWEV PRETLKLVERLGAGQFGEVW MGYYNGHTKV AVKSLKQGSM SPDAFLAEAN LMKQLQHQRLVRLYAVVTQE PIYIITEYME NGSLVDFLKT PSGIKLTINK LLDMAAQIAEGMAFIEERNY IHRDLRAANI LVSDTLSCKI ADFGLARLIE DNEYTAREGAKFPIKWTAPE AINYGTFTIK SDVWSFGILL TEIVTHGRIP YPGMTNPEVIQNLERGYRMV RPDNCPEELY QLMRLCWKER PEDRPTFDYL RSVLEDFFTATEGQFQPQPE QKLISEEDL SEQ ID NO: 87 vector clone: pIB1066 Sequence:MGHTRRQGTS PSKCPYLNFF QLLVLAGLSH FCSGEQKLIS EEDLVIHVTKEVKEVATLSC GHNVSVEELA QTRIYWQKEK KMVLTMMSGD MNIWPEYKNRTIFDITNNLS IVILALRPSD EGTYECVVLK YEKDAFKREH LAEVTLSVKADFPTPSISDF EIPTSNIRRI ICSTSGGFPE PHLSWLENGE ELNAINTTVSQDPETELYAV SSKLDFNMTT NHSFMCLIKY GHLRVNQTFN WNTTKQEHFPDNLLPSWAIT LISVNGIFVI CCLGCGCSSH PEDDWMENID VCENCHYPIVPLDGKGTLLI RNGSEVRDPL VTYEGSNPPA SPLQDNLVIA LHSYEPSHDGDLGFEKGEQL RILEQSGEWW KAQSLTTGQE GFIPFNFVAK ANSLEPEPWFFKNLSRKDAE RQLLAPGNTH GSFLIRESES TAGSFSLSVR DFDQNQGEVVKHYKIRNLDN GGFYISPRIT FPGLHELVRH YTNASDGLCT RLSRPCQTQKPQKPWWEDEW EVPRETLKLV ERLGAGQFGE VWMGYYNGHT KVAVKSLKQGSMSPDAFLAE ANLMKQLQHQ RLVRLYAVVT QEPIYIITEY MENGSLVDFLKTPSGIKLTI NKLLDMAAQI AEGMAFIEER NYIHRDLRAA NILVSDTLSCKIADFGLARL IEDNEYTARE GAKFPIKWTA PEAINYGTFT IKSDVWSFGILLTEIVTHGR IPYPGMTNPE VIQNLERGYR MVRPDNCPEE LYQLMRLCWKERPEDRPTFD YLRSVLEDFF TATEGQYQPQ P SEQ ID NO: 88 vector clone: pIB1067Sequence: MGHTRRQGTS PSKCPYLNFF QLLVLAGLSH FCSGEQKLIS EEDLVIHVTKEVKEVATLSC GHNVSVEELA QTRIYWQKEK KMVLTMMSGD MNIWPEYKNRTIFDITNNLS IVILALRPSD EGTYECVVLK YEKDAFKREH LAEVTLSVKADFPTPSISDF EIPTSNIRRI ICSTSGGFPE PHLSWLENGE ELNAINTTVSQDPETELYAV SSKLDFNMTT NHSFMCLIKY GHLRVNQTFN WNTTKQEHFPDNLLPSWAIT LISVNGIFVI CCLGCGCSSH PEDDWMENID VCENCHYPIVPLDGKGTLLI RNGSEVRDPL VTYEGSNPPA SPLQDNLVIA LHSYEPSHDGDLGFEKGEQL RILEQSGEWW KAQSLTTGQE GFIPFNFVAK ANSLEPEPWFFKNLSRKDAE RQLLAPGNTH GSFLIRESES TAGSFSLSVR DFDQNQGEVVKHYKIRNLDN GGFYISPRIT FPGLHELVRH YTNASDGLCT RLSRPCQTQKPQKPWWEDEW EVPRETLKLV ERLGAGQFGE VWMGYYNGHT KVAVKSLKQGSMSPDAFLAE ANLMKQLQHQ RLVRLYAVVT QEPIYIITEY MENGSLVDFLKTPSGIKLTI NKLLDMAAQI AEGMAFIEER NYIHRDLRAA NILVSDTLSCKIADFGLARL IEDNEYTARE GAKFPIKWTA PEAINYGTFT IKSDVWSFGILLTEIVTHGR IPYPGMTNPE VIQNLERGYR MVRPDNCPEE LYQLMRLCWKERPEDRPTFD YLRSVLEDFF TATEGQYQPQ PRSKRSRLLH SDYMNMTPRRPGPTRKHYQP YAPPRDFAAY RSKKVAKKPT NKAPHPKQEP QEINFPDDLPGSNTAAPVQE TLHGCQPVTQ EDGKESRISV QERQ SEQ ID NO: 89 vector clone:pIB1068 Sequence: MGHTRRQGTS PSKCPYLNFF QLLVLAGLSH FCSGEQKLIS EEDLVIHVTKEVKEVATLSC GHNVSVEELA QTRIYWQKEK KMVLTMMSGD MNIWPEYKNRTIFDITNNLS IVILALRPSD EGTYECVVLK YEKDAFKREH LAEVTLSVKADFPTPSISDF EIPTSNIRRI ICSTSGGFPE PHLSWLENGE ELNAINTTVSQDPETELYAV SSKLDFNMTT NHSFMCLIKY GHLRVNQTFN WNTTKQEHFPDNLLPSWAIT LISVNGIFVI CCLGCGCSSH PEDDWMENID VCENCHYPIVPLDGKGTLLI RNGSEVRDPL VTYEGSNPPA SPLQDNLVIA LHSYEPSHDGDLGFEKGEQL RILEQSGEWW KAQSLTTGQE GFIPFNFVAK ANSLEPEPWFFKNLSRKDAE RQLLAPGNTH GSFLIRESES TAGSFSLSVR DFDQNQGEVVKHYKIRNLDN GGFYISPRIT FPGLHELVRH YTNASDGLCT RLSRPCQTQKPQKPWWEDEW EVPRETLKLV ERLGAGQFGE VWMGYYNGHT KVAVKSLKQGSMSPDAFLAE ANLMKQLQHQ RLVRLYAVVT QEPIYIITEY MENGSLVDFLKTPSGIKLTI NKLLDMAAQI AEGMAFIEER NYIHRDLRAA NILVSDTLSCKIADFGLARL IEDNEYTARE GAKFPIKWTA PEAINYGTFT IKSDVWSFGILLTEIVTHGR IPYPGMTNPE VIQNLERGYR MVRPDNCPEE LYQLMRLCWKERPEDRPTFD YLRSVLEDFF TATEGQFQPQ P SEQ ID NO: 90 vector clone: pIB1069Sequence: MGHTRRQGTS PSKCPYLNFF QLLVLAGLSH FCSGEQKLIS EEDLVIHVTKEVKEVATLSC GHNVSVEELA QTRIYWQKEK KMVLTMMSGD MNIWPEYKNRTIFDITNNLS IVILALRPSD EGTYECVVLK YEKDAFKREH LAEVTLSVKADFPTPSISDF EIPTSNIRRI ICSTSGGFPE PHLSWLENGE ELNAINTTVSQDPETELYAV SSKLDFNMTT NHSFMCLIKY GHLRVNQTFN WNTTKQEHFPDNLLPSWAIT LISVNGIFVI CCLGCGCSSH PEDDWMENID VCENCHYPIVPLDGKGTLLI RNGSEVRDPL VTYEGSNPPA SPLQDNLVIA LHSYEPSHDGDLGFEKGEQL RILEQSGEWW KAQSLTTGQE GFIPFNFVAK ANSLEPEPWFFKNLSRKDAE RQLLAPGNTH GSFLIRESES TAGSFSLSVR DFDQNQGEVVKHYKIRNLDN GGFYISPRIT FPGLHELVRH YTNASDGLCT RLSRPCQTQKPQKPWWEDEW EVPRETLKLV ERLGAGQFGE VWMGYYNGHT KVAVKSLKQGSMSPDAFLAE ANLMKQLQHQ RLVRLYAVVT QEPIYIITEY MENGSLVDFLKTPSGIKLTI NKLLDMAAQI AEGMAFIEER NYIHRDLRAA NILVSDTLSCKIADFGLARL IEDNEYTARE GAKFPIKWTA PEAINYGTFT IKSDVWSFGILLTEIVTHGR IPYPGMTNPE VIQNLERGYR MVRPDNCPEE LYQLMRLCWKERPEDRPTFD YLRSVLEDFF TATEGQFQPQ PRSKRSRLLH SDYMNMTPRRPGPTRKHYQP YAPPRDFAAY RSKKVAKKPT NKAPHPKQEP QEINFPDDLPGSNTAAPVQE TLHGCQPVTQ EDGKESRISV QERQ SEQ ID NO: 91 vector clone:pIB1070 Sequence: MALPVTALLL PLALLLHAAR PEQKLISEED LMEEAILVPC VLGLLLLPILAMLMALCVHC HRLPGSYDST SSDSLYPRGI QFKRPHTVAP WPPAYPPVTSYPPLSQPDLL PIPRSPQPLG GSHRTPSSRR DSDGANSVAS YENEGASGIRGAQAGWGVWG PSWTRLTPVS LPPEPACEDA DEDEDDYHNP GYLVVLPDSTPATSTAAPSA PALSTPGIRD SAFSMESIDD YVNVPESGES AEASLDGSREYVNVSQELHP GAAKTEPAAL SSQEAEEVEE EGAPDYENLQ ELN SEQ ID NO: 92vector clone: pIB1071 Sequence:MALPVTALLL PLALLLHAAR PEQKLISEED LMEEAILVPC VLGLLLLPILAMLMALCVHC HRLPGSYDST SSDSLYPRGI QFKRPHTVAP WPPAYPPVTSYPPLSQPDLL PIPRSPQPLG GSHRTPSSRR DSDGANSVAS YENEGASGIRGAQAGWGVWG PSWTRLTPVS LPPEPACEDA DEDEDDYHNP GYLVVLPDSTPATSTAAPSA PALSTPGIRD SAFSMESIDD YVNVPESGES AEASLDGSREYVNVSQELHP GAAKTEPAAL SSQEAEEVEE EGAPDYENLQ ELNRSKRSRLLHSDYMNMTP RRPGPTRKHY QPYAPPRDFA AYRSKKVAKK PTNKAPHPKQEPQEINFPDD LPGSNTAAPV QETLHGCQPV TQEDGKESRI SVQERQ SEQ ID NO: 93vector clone: pIB1072 Sequence:MNRGVPFRHL LLVLQLALLP AATQGEQKLI SEEDLKKVVL GKKGDTVELTCTASQKKSIQ FHWKNSNQIK ILGNQGSFLT KGPSKLNDRA DSRRSLWDQGNFPLIIKNLK IEDSDTYICE VEDQKEEVQL LVFGLTANSD THLLQGQSLTLTLESPPGSS PSVQCRSPRG KNIQGGKTLS VSQLELQDSG TWTCTVLQNQKKVEFKIDIV VLAFQKASSI VYKKEGEQVE FSFPLAFTVE KLTGSGELWWQAERASSSKS WITFDLKNKE VSVKRVTQDP KLQMGKKLPL HLTLPQALPQYAGSGNLTLA LEAKTGKLHQ EVNLVVMRAT QLQKNLTCEV WGPTSPKLMLSLKLENKEAK VSKREKAVWV LNPEAGMWQC LLSDSGQVLL ESNIKVLPTWSTPVQPMALI VLGGVAGLLL FIGLGIFFCV RCRHRRRQAE RMSQIKRLLSEKKTCQCPHR FQKTCSPI SEQ ID NO: 94 vector clone: pIB1073 Sequence:MNRGVPFRHL LLVLQLALLP AATQGEQKLI SEEDLKKVVL GKKGDTVELTCTASQKKSIQ FHWKNSNQIK ILGNQGSFLT KGPSKLNDRA DSRRSLWDQGNFPLIIKNLK IEDSDTYICE VEDQKEEVQL LVFGLTANSD THLLQGQSLTLTLESPPGSS PSVQCRSPRG KNIQGGKTLS VSQLELQDSG TWTCTVLQNQKKVEFKIDIV VLAFQKASSI VYKKEGEQVE FSFPLAFTVE KLTGSGELWWQAERASSSKS WITFDLKNKE VSVKRVTQDP KLQMGKKLPL HLTLPQALPQYAGSGNLTLA LEAKTGKLHQ EVNLVVMRAT QLQKNLTCEV WGPTSPKLMLSLKLENKEAK VSKREKAVWV LNPEAGMWQC LLSDSGQVLL ESNIKVLPTWSTPVQPMALI VLGGVAGLLL FIGLGIFFCV RCRHRRRQAE RMSQIKRLLSEKKTCQCPHR FQKTCSPIRS KRSRLLHSDY MNMTPRRPGP TRKHYQPYAPPRDFAAYRSK KVAKKPTNKA PHPKQEPQEI NFPDDLPGSN TAAPVQETLHGCQPVTQEDG KESRISVQER Q SEQ ID NO: 95 vector clone: pIB1074 Sequence:MALPVTALLL PLALLLHAAR PEQKLISEED LSQFRVSPLD RTWNLGETVELKCQVLLSNP TSGCSWLFQP RGAAASPTFL LYLSQNKPKA AEGLDTQRFSGKRLGDTFVL TLSDFRRENE GYYFCSALSN SIMYFSHFVP VFLPAKPTTTPAPRPPTPAP TIASQPLSLR PEACRPAAGG AVHTRGLDFA CDIYIWAPLAGTCGVLLLSL VITLYCNHRN RRRVCKCPRP VVKSGDKPSL SARYVRAKRGSGEGRGSLLT CGDVEENPGP MRPRLWLLLA AQLTVLHGNS VDYKDDDDKLQQTPAYIKVQ TNKMVMLSCE AKISLSNMRI YWLRQRQAPS SDSHHEFLALWDSAKGTIHG EEVEQEKIAV FRDASRFILN LTSVKPEDSG IYFCMIVGSPELTFGKGTQL SVVDFLPTTA QPTKKSTLKK RVCRLPRPET QKGPLCSPITLGLLVAGVLV LLVSLGVAIH LCCRRRRARL RFMKQFYK SEQ ID NO: 96 vector clone:pIB1075 Sequence: MALPVTALLL PLALLLHAAR PEQKLISEED LSQFRVSPLD RTWNLGETVELKCQVLLSNP TSGCSWLFQP RGAAASPTFL LYLSQNKPKA AEGLDTQRFSGKRLGDTFVL TLSDFRRENE GYYFCSALSN SIMYFSHFVP VFLPAKPTTTPAPRPPTPAP TIASQPLSLR PEACRPAAGG AVHTRGLDFA CDIYIWAPLAGTCGVLLLSL VITLYCNHRN RRRVCKCPRP VVKSGDKPSL SARYVRSKRSRLLHSDYMNM TPRRPGPTRK HYQPYAPPRD FAAYRSKKVA KKPTNKAPHPKQEPQEINFP DDLPGSNTAA PVQETLHGCQ PVTQEDGKES RISVQERQRAKRGSGEGRGS LLTCGDVEEN PGPMRPRLWL LLAAQLTVLH GNSVDYKDDDDKLQQTPAYI KVQTNKMVML SCEAKISLSN MRIYWLRQRQ APSSDSHHEFLALWDSAKGT IHGEEVEQEK IAVFRDASRF ILNLTSVKPE DSGIYFCMIVGSPELTFGKG TQLSVVDFLP TTAQPTKKST LKKRVCRLPR PETQKGPLCSPITLGLLVAG VLVLLVSLGV AIHLCCRRRR ARLRFMKQFY KRSKRSRLLHSDYMNMTPRR PGPTRKHYQP YAPPRDFAAY RSKKVAKKPT NKAPHPKQEPQEINFPDDLP GSNTAAPVQE TLHGCQPVTQ EDGKESRISV QERQ SEQ ID NO: 97vector clone: pIB1076 Sequence:MALPVTALLL PLALLLHAAR PEQKLISEED LELTDTLQAE TDQLEDEKSALQTEIANLLK EKEKLEFILA AHNCTYGCTG PGLEGCPTNG PKIPSIATGMVGALLLLLVV ALGIGLFMRS KRSRLLHSDY MNMTPRRPGP TRKHYQPYAPPRDFAAYRSK KVAKKPTNKA PHPKQEPQEI NFPDDLPGSN TAAPVQETLHGCQPVTQEDG KESRISVQER Q SEQ ID NO: 98 vector clone: pIB1077 Sequence:MALPVTALLL PLALLLHAAR PEQKLISEED LELTDTLQAE TDQLEDEKSALQTEIANLLK EKEKLEFILA AHFWVLVVVG GVLACYSLLV TVAFIIFWVRSKRSRLLHSD YMNMTPRRPG PTRKHYQPYA PPRDFAAYRS KKVAKKPTNKAPHPKQEPQE INFPDDLPGS NTAAPVQETL HGCQPVTQED GKESRISVQE RQ SEQ ID NO: 99vector clone: pIB1078 Sequence:MALPVTALLL PLALLLHAAR PEQKLISEED LLEEKVKTLK AQNSELASTANMLREQVAQL NCTYGCTGPG LEGCPTNGPK IPSIATGMVG ALLLLLVVALGIGLFMRSKR SRLLHSDYMN MTPRRPGPTR KHYQPYAPPR DFAAYRSKKVAKKPTNKAPH PKQEPQEINF PDDLPGSNTA APVQETLHGC QPVTQEDGKE SRISVQERQSEQ ID NO: 100 vector clone: pIB1079 Sequence:MALPVTALLL PLALLLHAAR PEQKLISEED LLEEKVKTLK AQNSELASTANMLREQVAQL FWVLVVVGGV LACYSLLVTV AFIIFWVRSK RSRLLHSDYMNMTPRRPGPT RKHYQPYAPP RDFAAYRSKK VAKKPTNKAP HPKQEPQEINFPDDLPGSNT AAPVQETLHG CQPVTQEDGK ESRISVQERQ SEQ ID NO: 101 vector clone:pIB1080 Sequence: MALPVTALLL PLALLLHAAR PEQKLISEED LETQHKVLEL TAENERLQKKVEQLSRELST NCTYGCTGPG LEGCPTNGPK IPSIATGMVG ALLLLLVVALGIGLFMRSKR SRLLHSDYMN MTPRRPGPTR KHYQPYAPPR DFAAYRSKKVAKKPTNKAPH PKQEPQEINF PDDLPGSNTA APVQETLHGC QPVTQEDGKE SRISVQERQSEQ ID NO: 102 vector clone: pIB1081 Sequence:MALPVTALLL PLALLLHAAR PEQKLISEED LETQHKVLEL TAENERLQKKVEQLSRELST FWVLVVVGGV LACYSLLVTV AFIIFWVRSK RSRLLHSDYMNMTPRRPGPT RKHYQPYAPP RDFAAYRSKK VAKKPTNKAP HPKQEPQEINFPDDLPGSNT AAPVQETLHG CQPVTQEDGK ESRISVQERQ SEQ ID NO: 103 vector clone:pIB1082 Sequence: MRPSGTAGAA LLALLAALCP ASRAEQKLIS EEDLLEEKKV CQGTSNKLTQLGTFEDHFLS LQRMFNNCEV VLGNLEITYV QRNYDLSFLK TIQEVAGYVLIALNTVERIP LENLQIIRGN MYYENSYALA VLSNYDANKT GLKELPMRNLQEILHGAVRF SNNPALCNVE SIQWRDIVSS DFLSNMSMDF QNHLGSCQKCDPSCPNGSCW GAGEENCQKL TKIICAQQCS GRCRGKSPSD CCHNQCAAGCTGPRESDCLV CRKFRDEATC KDTCPPLMLY NPTTYQMDVN PEGKYSFGATCVKKCPRNYV VTDHGSCVRA CGADSYEMEE DGVRKCKKCE GPCRKVCNGIGIGEFKDSLS INATNIKHFK NCTSISGDLH ILPVAFRGDS FTHTPPLDPQELDILKTVKE ITGFLLIQAW PENRTDLHAF ENLEIIRGRT KQHGQFSLAVVSLNITSLGL RSLKEISDGD VIISGNKNLC YANTINWKKL FGTSGQKTKIISNRGENSCK ATGQVCHALC SPEGCWGPEP RDCVSCRNVS RGRECVDKCNLLEGEPREFV ENSECIQCHP ECLPQAMNIT CTGRGPDNCI QCAHYIDGPHCVKTCPAGVM GENNTLVWKY ADAGHVCHLC HPNCTYGCTG PGLEGCPTNGPKIPSIATGM VGALLLLLVV ALGIGLFMRS KRSRLLHSDY MNMTPRRPGPTRKHYQPYAP PRDFAAYRSK KVAKKPTNKA PHPKQEPQEI NFPDDLPGSNTAAPVQETLH GCQPVTQEDG KESRISVQER Q SEQ ID NO: 104 vector clone: pIB1083Sequence: MRPSGTAGAA LLALLAALCP ASRAEQKLIS EEDLGTSGQK TKIISNRGENSCKATGQVCH ALCSPEGCWG PEPRDCVSCR NVSRGRECVD KCNLLEGEPREFVENSECIQ CHPECLPQAM NITCTGRGPD NCIQCAHYID GPHCVKTCPAGVMGENNTLV WKYADAGHVC HLCHPNCTYG CTGPGLEGCP TNGPKIPSIATGMVGALLLL LVVALGIGLF MRSKRSRLLH SDYMNMTPRR PGPTRKHYQPYAPPRDFAAY RSKKVAKKPT NKAPHPKQEP QEINFPDDLP GSNTAAPVQETLHGCQPVTQ EDGKESRISV QERQ SEQ ID NO: 105 vector clone: pIB1084Sequence: MRPSGTAGAA LLALLAALCP ASRAEQKLIS EEDLNCTYGC TGPGLEGCPTNGPKIPSIAT GMVGALLLLL VVALGIGLFM RSKRSRLLHS DYMNMTPRRPGPTRKHYQPY APPRDFAAYR SKKVAKKPTN KAPHPKQEPQ EINFPDDLPGSNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ SEQ ID NO: 106 vector clone:pIB1085 Sequence: MELAALCRWG LLLALLPPGA ASEQKLISEE DLTQVCTGTD MKLRLPASPETHLDMLRHLY QGCQVVQGNL ELTYLPTNAS LSFLQDIQEV QGYVLIAHNQVRQVPLQRLR IVRGTQLFED NYALAVLDNG DPLNNTTPVT GASPGGLRELQLRSLTEILK GGVLIQRNPQ LCYQDTILWK DIFHKNNQLA LTLIDTNRSRACHPCSPMCK GSRCWGESSE DCQSLTRTVC AGGCARCKGP LPTDCCHEQCAAGCTGPKHS DCLACLHFNH SGICELHCPA LVTYNTDTFE SMPNPEGRYTFGASCVTACP YNYLSTDVGS CTLVCPLHNQ EVTAEDGTQR CEKCSKPCARVCYGLGMEHL REVRAVTSAN IQEFAGCKKI FGSLAFLPES FDGDPASNTAPLQPEQLQVF ETLEEITGYL YISAWPDSLP DLSVFQNLQV IRGRILHNGAYSLTLQGLGI SWLGLRSLRE LGSGLALIHH NTHLCFVHTV PWDQLFRNPHQALLHTANRP EDECVGEGLA CHQLCARGHC WGPGPTQCVN CSQFLRGQECVEECRVLQGL PREYVNARHC LPCHPECQPQ NGSVTCFGPE ADQCVACAHYKDPPFCVARC PSGVKPDLSY MPIWKFPDEE GACQPCPINC THSCVDLDDKGCPAEQRASP LTSIISAVVG ILLVVVLGVV FGILIRSKRS RLLHSDYMNMTPRRPGPTRK HYQPYAPPRD FAAYRSKKVA KKPTNKAPHP KQEPQEINFPDDLPGSNTAA PVQETLHGCQ PVTQEDGKES RISVQERQ SEQ ID NO: 107 vector clone:pIB1086 Sequence: MELAALCRWG LLLALLPPGA ASEQKLISEE DLTQVCTGTD MKLRLPASPETHLDMLRHLY QGCQVVQGNL ELTYLPTNAS LSFLQDIQEV QGYVLIAHNQVRQVPLQRLR IVRGTQLFED NYALAVLDNG DPLNNTTPVT GASPGGLRELQLRSLTEILK GGVLIQRNPQ LCYQDTILWK DIFHKNNQLA LTLIDTNRSRACHPCSPMCK GSRCWGESSE DCQSLTRTVC AGGCARCKGP LPTDCCHEQCAAGCTGPKHS DCLACLHFNH SGICELHCPA LVTYNTDTFE SMPNPEGRYTFGASCVTACP YNYLSTDVGS CTLVCPLHNQ EVTAEDGTQR CEKCSKPCARVCYGLGMEHL REVRAVTSAN IQEFAGCKKI FGSLAFLPES FDGDPASNTAPLQPEQLQVF ETLEEITGYL YISAWPDSLP DLSVFQNLQV IRGRILHNGAYSLTLQGLGI SWLGLRSLRE LGSGLALIHH NTHLCFVHTV PWDQLFRNPHQALLHTANRP EDECVGEGLA CHQLCARGHC WGPGPTQCVN CSQFLRGQECVEECRVLQGL PREYVNARHC LPCHPECQPQ NGSVTCFGPE ADQCVACAHYKDPPFCVARC PSGVKPDLSY MPIWKFPDEE GACQPCPINC THSCVDLDDKGCPAEQRASP LTSIISAVEG ILLVVVLGVV FGILIRSKRS RLLHSDYMNMTPRRPGPTRK HYQPYAPPRD FAAYRSKKVA KKPTNKAPHP KQEPQEINFPDDLPGSNTAA PVQETLHGCQ PVTQEDGKES RISVQERQ SEQ ID NO: 108 vector clone:pIB1087 Sequence: MELAALCRWG LLLALLPPGA ASEQKLISEE DLTQVCTGTD MKLRLPASPETHLDMLRHLY QGCQVVQGNL ELTYLPTNAS LSFLQDIQEV QGYVLIAHNQVRQVPLQRLR IVRGTQLFED NYALAVLDNG DPLNNTTPVT GASPGGLRELQLRSLTEILK GGVLIQRNPQ LCYQDTILWK DIFHKNNQLA LTLIDTNRSRACHPCSPMCK GSRCWGESSE DCQSLTRTVC AGGCARCKGP LPTDCCHEQCAAGCTGPKHS DCLACLHFNH SGICELHCPA LVTYNTDTFE SMPNPEGRYTFGASCVTACP YNYLSTDVGS CTLVCPLHNQ EVTAEDGTQR CEKCSKPCARVCYGLGMEHL REVRAVTSAN IQEFAGCKKI FGSLAFLPES FDGDPASNTAPLQPEQLQVF ETLEEITGYL YISAWPDSLP DLSVFQNLQV IRGRILHNGAYSLTLQGLGI SWLGLRSLRE LGSGLALIHH NTHLCFVHTV PWDQLFRNPHQALLHTANRP EDECVGEGLA CHQLCARGHC WGPGPTQCVN CSQFLRGQECVEECRVLQGL PREYVNARHC LPCHPECQPQ NGSVTCFGPE ADQCVACAHYKDPPFCVARC PSGVKPDLSY MPIWKFPDEE GACQPCPINC THSCVDLDDKGCPAEQRASP LTSIISAVVD ILLVVVLGVV FGILIRSKRS RLLHSDYMNMTPRRPGPTRK HYQPYAPPRD FAAYRSKKVA KKPTNKAPHP KQEPQEINFPDDLPGSNTAA PVQETLHGCQ PVTQEDGKES RISVQERQ SEQ ID NO: 109 vector clone:pIB1088 Sequence: MELAALCRWG LLLALLPPGA ASEQKLISEE DLTQVCTGTD MKLRLPASPETHLDMLRHLY QGCQVVQGNL ELTYLPTNAS LSFLQDIQEV QGYVLIAHNQVRQVPLQRLR IVRGTQLFED NYALAVLDNG DPLNNTTPVT GASPGGLRELQLRSLTEILK GGVLIQRNPQ LCYQDTILWK DIFHKNNQLA LTLIDTNRSRACHPCSPMCK GSRCWGESSE DCQSLTRTVC AGGCARCKGP LPTDCCHEQCAAGCTGPKHS DCLACLHFNH SGICELHCPA LVTYNTDTFE SMPNPEGRYTFGASCVTACP YNYLSTDVGS CTLVCPLHNQ EVTAEDGTQR CEKCSKPCARVCYGLGMEHL REVRAVTSAN IQEFAGCKKI FGSLAFLPES FDGDPASNTAPLQPEQLQVF ETLEEITGYL YISAWPDSLP DLSVFQNLQV IRGRILHNGAYSLTLQGLGI SWLGLRSLRE LGSGLALIHH NTHLCFVHTV PWDQLFRNPHQALLHTANRP EDECVGEGLA CHQLCARGHC WGPGPTQCVN CSQFLRGQECVEECRVLQGL PREYVNARHC LPCHPECQPQ NGSVTCFGPE ADQCVACAHYKDPPFCVARC PSGVKPDLSY MPIWKFPDEE GACQPCPINC THSCVDLDDKGCPAEQRASP LTSIISAVVR ILLVVVLGVV FGILIRSKRS RLLHSDYMNMTPRRPGPTRK HYQPYAPPRD FAAYRSKKVA KKPTNKAPHP KQEPQEINFPDDLPGSNTAA PVQETLHGCQ PVTQEDGKES RISVQERQ SEQ ID NO: 110 vector clone:pIB1089 Sequence: MELAALCRWG LLLALLPPGA ASEQKLISEE DLQLCARGHC WGPGPTQCVNCSQFLRGQEC VEECRVLQGL PREYVNARHC LPCHPECQPQ NGSVTCFGPEADQCVACAHY KDPPFCVARC PSGVKPDLSY MPIWKFPDEE GACQPCPINCTHSCVDLDDK GCPAEQRASP LTSIISAVVG ILLVVVLGVV FGILIRSKRSRLLHSDYMNM TPRRPGPTRK HYQPYAPPRD FAAYRSKKVA KKPTNKAPHPKQEPQEINFP DDLPGSNTAA PVQETLHGCQ PVTQEDGKES RISVQERQ SEQ ID NO: 111vector clone: pIB1090 Sequence:MELAALCRWG LLLALLPPGA ASEQKLISEE DLQLCARGHC WGPGPTQCVNCSQFLRGQEC VEECRVLQGL PREYVNARHC LPCHPECQPQ NGSVTCFGPEADQCVACAHY KDPPFCVARC PSGVKPDLSY MPIWKFPDEE GACQPCPINCTHSCVDLDDK GCPAEQRASP LTSIISAVEG ILLVVVLGVV FGILIRSKRSRLLHSDYMNM TPRRPGPTRK HYQPYAPPRD FAAYRSKKVA KKPTNKAPHPKQEPQEINFP DDLPGSNTAA PVQETLHGCQ PVTQEDGKES RISVQERQ SEQ ID NO: 112vector clone: pIB1091 Sequence:MELAALCRWG LLLALLPPGA ASEQKLISEE DLQLCARGHC WGPGPTQCVNCSQFLRGQEC VEECRVLQGL PREYVNARHC LPCHPECQPQ NGSVTCFGPEADQCVACAHY KDPPFCVARC PSGVKPDLSY MPIWKFPDEE GACQPCPINCTHSCVDLDDK GCPAEQRASP LTSIISAVVD ILLVVVLGVV FGILIRSKRSRLLHSDYMNM TPRRPGPTRK HYQPYAPPRD FAAYRSKKVA KKPTNKAPHPKQEPQEINFP DDLPGSNTAA PVQETLHGCQ PVTQEDGKES RISVQERQ SEQ ID NO: 113vector clone: pIB1092 Sequence:MELAALCRWG LLLALLPPGA ASEQKLISEE DLQLCARGHC WGPGPTQCVNCSQFLRGQEC VEECRVLQGL PREYVNARHC LPCHPECQPQ NGSVTCFGPEADQCVACAHY KDPPFCVARC PSGVKPDLSY MPIWKFPDEE GACQPCPINCTHSCVDLDDK GCPAEQRASP LTSIISAVVR ILLVVVLGVV FGILIRSKRSRLLHSDYMNM TPRRPGPTRK HYQPYAPPRD FAAYRSKKVA KKPTNKAPHPKQEPQEINFP DDLPGSNTAA PVQETLHGCQ PVTQEDGKES RISVQERQ SEQ ID NO: 114vector clone: pIB1093 Sequence:MELAALCRWG LLLALLPPGA ASEQKLISEE DLSIISAVVG ILLVVVLGVVFGILIRSKRS RLLHSDYMNM TPRRPGPTRK HYQPYAPPRD FAAYRSKKVAKKPTNKAPHP KQEPQEINFP DDLPGSNTAA PVQETLHGCQ PVTQEDGKES RISVQERQSEQ ID NO: 115 vector clone: pIB1094 Sequence:MELAALCRWG LLLALLPPGA ASEQKLISEE DLSIISAVEG ILLVVVLGVVFGILIRSKRS RLLHSDYMNM TPRRPGPTRK HYQPYAPPRD FAAYRSKKVAKKPTNKAPHP KQEPQEINFP DDLPGSNTAA PVQETLHGCQ PVTQEDGKES RISVQERQSEQ ID NO: 116 vector clone: pIB1095 Sequence:MELAALCRWG LLLALLPPGA ASEQKLISEE DLSIISAVVD ILLVVVLGVVFGILIRSKRS RLLHSDYMNM TPRRPGPTRK HYQPYAPPRD FAAYRSKKVAKKPTNKAPHP KQEPQEINFP DDLPGSNTAA PVQETLHGCQ PVTQEDGKES RISVQERQSEQ ID NO: 117 vector clone: pIB1096 Sequence:MELAALCRWG LLLALLPPGA ASEQKLISEE DLSIISAVVR ILLVVVLGVVFGILIRSKRS RLLHSDYMNM TPRRPGPTRK HYQPYAPPRD FAAYRSKKVAKKPTNKAPHP KQEPQEINFP DDLPGSNTAA PVQETLHGCQ PVTQEDGKES RISVQERQSEQ ID NO: 118 vector clone: pIB1097 Sequence:MALPVTALLL PLALLLHAAR PEQKLISEED LQEQLVESGG RLVTPGTPLTLTCTASGFSL GSDFMSWVRQ APGKGLEWIG YIDPRSDIPY YASWAKGRFTISKTSTTVDL KITSPTTEDT ATYFCARDLN AGYFNGIFYI WGPGTLVTVSSGGGGSGGGG SGGGGSELVM TQTPSSVSAA VGDTVTINCQ ASETVATLLAWYQQKPGQPP KLLIYGASNL ESGVPSRFRG SGSGTEFTLT ISGMKAEDAATYYCQYGYIS TGSNTFGAGT NVEIKAAAGS GGSGILVKQS PMLVAYDNAVNLSCKYSYNL FSREFRASLH KGLDSAVEVC VVYGNYSQQL QVYSKTGFNCDGKLGNESVT FYLQNLYVNQ TDIYFCKIEV MYPPPYLDNE KSNGTIIHVKGKHLCPSPLF PGPSKPFWVL VVVGGVLACY SLLVTVAFII FWVRSKRSRLLHSDYMNMTP RRPGPTRKHY QPYAPPRDFA AYRSKKVAKK PTNKAPHPKQEPQEINFPDD LPGSNTAAPV QETLHGCQPV TQEDGKESRI SVQERQ SEQ ID NO: 119vector clone: pIB1098 Sequence:MALPVTALLL PLALLLHAAR PEQKLISEED LELVMTQTPS SVSAAVGDTVTINCQASETV ATLLAWYQQK PGQPPKLLIY GASNLESGVP SRFRGSGSGTEFTLTISGMK AEDAATYYCQ YGYISTGSNT FGAGTNVEIK GGGGSGGGGSGGGGSQEQLV ESGGRLVTPG TPLTLTCTAS GFSLGSDFMS WVRQAPGKGLEWIGYIDPRS DIPYYASWAK GRFTISKTST TVDLKITSPT TEDTATYFCARDLNAGYFNG IFYIWGPGTL VTVSSAAAGS GGSGILVKQS PMLVAYDNAVNLSCKYSYNL FSREFRASLH KGLDSAVEVC VVYGNYSQQL QVYSKTGFNCDGKLGNESVT FYLQNLYVNQ TDIYFCKIEV MYPPPYLDNE KSNGTIIHVKGKHLCPSPLF PGPSKPFWVL VVVGGVLACY SLLVTVAFII FWVRSKRSRLLHSDYMNMTP RRPGPTRKHY QPYAPPRDFA AYRSKKVAKK PTNKAPHPKQEPQEINFPDD LPGSNTAAPV QETLHGCQPV TQEDGKESRI SVQERQ SEQ ID NO: 120vector clone: pIB1099 Sequence:MALPVTALLL PLALLLHAAR PEQKLISEED LQEQLVESGG RLVTPGTPLTLTCTASGFSL GSDFMSWVRQ APGKGLEWIG YIDPRSDIPY YASWAKGRFTISKTSTTVDL KITSPTTEDT ATYFCARDLN AGYFNGIFYI WGPGTLVTVSSGGGGSGGGG SGGGGSELDM TQTPSSTSEP VGGTVTINCQ ASQTISSYLSWYQQKPGHPP KLLIYDASDL ASGVPSRFSG SRSGTQFTLT ISGVQCDDAATYYCLGVYDY RSDDGAAFGG GTELEILAAA GSGGSGILVK QSPMLVAYDNAVNLSCKYSY NLFSREFRAS LHKGLDSAVE VCVVYGNYSQ QLQVYSKTGFNCDGKLGNES VTFYLQNLYV NQTDIYFCKI EVMYPPPYLD NEKSNGTIIHVKGKHLCPSP LFPGPSKPFW VLVVVGGVLA CYSLLVTVAF IIFWVRSKRSRLLHSDYMNM TPRRPGPTRK HYQPYAPPRD FAAYRSKKVA KKPTNKAPHPKQEPQEINFP DDLPGSNTAA PVQETLHGCQ PVTQEDGKES RISVQERQ SEQ ID NO: 121vector clone: pIB1100 Sequence:MALPVTALLL PLALLLHAAR PEQKLISEED LELDMTQTPS STSEPVGGTVTINCQASQTI SSYLSWYQQK PGHPPKLLIY DASDLASGVP SRFSGSRSGTQFTLTISGVQ CDDAATYYCL GVYDYRSDDG AAFGGGTELE ILGGGGSGGGGSGGGGSQEQ LVESGGRLVT PGTPLTLTCT ASGFSLGSDF MSWVRQAPGKGLEWIGYIDP RSDIPYYASW AKGRFTISKT STTVDLKITS PTTEDTATYFCARDLNAGYF NGIFYIWGPG TLVTVSSAAA GSGGSGILVK QSPMLVAYDNAVNLSCKYSY NLFSREFRAS LHKGLDSAVE VCVVYGNYSQ QLQVYSKTGFNCDGKLGNES VTFYLQNLYV NQTDIYFCKI EVMYPPPYLD NEKSNGTIIHVKGKHLCPSP LFPGPSKPFW VLVVVGGVLA CYSLLVTVAF IIFWVRSKRSRLLHSDYMNM TPRRPGPTRK HYQPYAPPRD FAAYRSKKVA KKPTNKAPHPKQEPQEINFP DDLPGSNTAA PVQETLHGCQ PVTQEDGKES RISVQERQ SEQ ID NO: 122vector clone: pIB1101 Sequence:MALPVTALLL PLALLLHAAR PEQKLISEED LQSLEESGGR LVTPGTPLTLTCTVSGFSLS TNDMNWVRQA PGKGLEWIGV IYSDDTPDYA TWAKGRFTISRTSTTVDLKI TSPTTEDTAT YFCARGHYDS AVYAYALNIW GPGTLVTVSSGGGGSGGGGS GGGGSELVMT QTPSSVSAAV GGTVTITCQA SQSLSNLLAWYQQKPGQPPK LLIYGASNLE SGVPSRFRGS GSGTDFTLTI SGMKAEDAATYYCQGGHYSG LTFGNGTNVE IKAAAGSGGS GILVKQSPML VAYDNAVNLSCKYSYNLFSR EFRASLHKGL DSAVEVCVVY GNYSQQLQVY SKTGFNCDGKLGNESVTFYL QNLYVNQTDI YFCKIEVMYP PPYLDNEKSN GTIIHVKGKHLCPSPLFPGP SKPFWVLVVV GGVLACYSLL VTVAFIIFWV RSKRSRLLHSDYMNMTPRRP GPTRKHYQPY APPRDFAAYR SKKVAKKPTN KAPHPKQEPQEINFPDDLPG SNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ SEQ ID NO: 123vector clone: pIB1102 Sequence:MALPVTALLL PLALLLHAAR PEQKLISEED LELVMTQTPS SVSAAVGGTVTITCQASQSL SNLLAWYQQK PGQPPKLLIY GASNLESGVP SRFRGSGSGTDFTLTISGMK AEDAATYYCQ GGHYSGLTFG NGTNVEIKGG GGSGGGGSGGGGSQSLEESG GRLVTPGTPL TLTCTVSGFS LSTNDMNWVR QAPGKGLEWIGVIYSDDTPD YATWAKGRFT ISRTSTTVDL KITSPTTEDT ATYFCARGHYDSAVYAYALN IWGPGTLVTV SSAAAGSGGS GILVKQSPML VAYDNAVNLSCKYSYNLFSR EFRASLHKGL DSAVEVCVVY GNYSQQLQVY SKTGFNCDGKLGNESVTFYL QNLYVNQTDI YFCKIEVMYP PPYLDNEKSN GTIIHVKGKHLCPSPLFPGP SKPFWVLVVV GGVLACYSLL VTVAFIIFWV RSKRSRLLHSDYMNMTPRRP GPTRKHYQPY APPRDFAAYR SKKVAKKPTN KAPHPKQEPQEINFPDDLPG SNTAAPVQET LHGCQPVTQE DGKESRISVQ ERQ SEQ ID NO: 124vector clone: pIB1103 Sequence:MYGKIIFVLL LSEIVSISAE QKLISEEDLS STTGVAMHTS TSSSVTKSYISSQTNDTHKR DTYAATPRAH EVSEISVRTV YPPEEETGER VQLAHHFSEPEITLIIFGVM AGVIGTILLI SYGRSKRSRL LHSDYMNMTP RRPGPTRKHYQPYAPPRDFA AYRSKKVAKK PTNKAPHPKQ EPQEINFPDD LPGSNTAAPVQETLHGCQPV TQEDGKESRI SVQERQ SEQ ID NO: 125 vector clone: pIB1104Sequence: MYGKIIFVLL LSEIVSISAE QKLISEEDLI TLIIFGVMAG VIGTILLISYGRSKRSRLLH SDYMNMTPRR PGPTRKHYQP YAPPRDFAAY RSKKVAKKPTNKAPHPKQEP QEINFPDDLP GSNTAAPVQE TLHGCQPVTQ EDGKESRISV QERQSEQ ID NO: 126 vector clone: pIB1105 Sequence:MDHLGASLWP QVGSLCLLLA GAAWEQKLIS EEDLAPPPNL PDPKFESKAALLAARGPEEL LCFTERLEDL VCFWEEAASA GVGPGNYSFS YQLEDEPWKLCRLHQAPTAR GAVRFWCSLP TADTSSFVPL ELRVTAASGA PRYHRVIHINEVVLLDAPVG LVARLADESG HVVLRWLPPP ETPMTSHIRY EVDVSAGNGAGSVQRVEILE GRTECVLSNL RGRTRYTFAV RARMAEPSFG GFWSAWSEPVSLLTPSDLDP LILTLSLILV VILVLLTVLA LLSRSKRSRL LHSDYMNMTPRRPGPTRKHY QPYAPPRDFA AYRSKKVAKK PTNKAPHPKQ EPQEINFPDDLPGSNTAAPV QETLHGCQPV TQEDGKESRI SVQERQ SEQ ID NO: 127 vector clone:pIB1106 Sequence: MDHLGASLWP QVGSLCLLLA GAAWEQKLIS EEDLLILTLS LILVVILVLLTVLALLSRSK RSRLLHSDYM NMTPRRPGPT RKHYQPYAPP RDFAAYRSKKVAKKPTNKAP HPKQEPQEIN FPDDLPGSNT AAPVQETLHG CQPVTQEDGK ESRISVQERQSEQ ID NO: 128 vector clone: pIB1107 Sequence:MPSWALFMVT SCLLLAPQNL AQVSSEQKLI SEEDLQDVSL LASDSEPLKCFSRTFEDLTC FWDEEEAAPS GTYQLLYAYP REKPRACPLS SQSMPHFGTRYVCQFPDQEE VRLFFPLHLW VKNVFLNQTR TQRVLFVDSV GLPAPPSIIKAMGGSQPGEL QISWEEPAPE ISDFLRYELR YGPRDPKNST GPTVIQLIATETCCPALQRP HSASALDQSP CAQPTMPWQD GPKQTSPSRE ASALTAEGGSCLISGLQPGN SYWLQLRSEP DGISLGGSWG SWSLPVTVDL PGDAVALGLQCFTLDLKNVT CQWQQQDHAS SQGFFYHSRA RCCPRDRYPI WENCEEEEKTNPGLQTPQFS RCHFKSRNDS IIHILVEVTT APGTVHSYLG SPFWIHQAVRLPTPNLHWRE ISSGHLELEW QHPSSWAAQE TCYQLRYTGE GHQDWKVLEPPLGARGGTLE LRPRSRYRLQ LRARLNGPTY QGPWSSWSDP TRVETATETAWISLVTALHL VLGLSAVLGL LLLRWRSKRS RLLHSDYMNM TPRRPGPTRKHYQPYAPPRD FAAYRSKKVA KKPTNKAPHP KQEPQEINFP DDLPGSNTAAPVQETLHGCQ PVTQEDGKES RISVQERQ SEQ ID NO: 129 vector clone: pIB1108Sequence: MPSWALFMVT SCLLLAPQNL AQVSSEQKLI SEEDLISLVT ALHLVLGLSAVLGLLLLRWR SKRSRLLHSD YMNMTPRRPG PTRKHYQPYA PPRDFAAYRSKKVAKKPTNK APHPKQEPQE INFPDDLPGS NTAAPVQETL HGCQPVTQED GKESRISVQE RQSEQ ID NO: 130 vector clone: pIB1109 Sequence:MPSWALFMVT SCLLLAPQNL AQVSSEQKLI SEEDLQDVSL LASDSEPLKCFSRTFEDLTC FWDEEEAAPS GTYQLLYAYP REKPRACPLS SQSMPHFGTRYVCQFPDQEE VRLFFPLHLW VKNVFLNQTR TQRVLFVDSV GLPAPPSIIKAMGGSQPGEL QISWEEPAPE ISDFLRYELR YGPRDPKNST GPTVIQLIATETCCPALQRP HSASALDQSP CAQPTMPWQD GPKQTSPSRE ASALTAEGGSCLISGLQPGN SYWLQLRSEP DGISLGGSWG SWSLPVTVDL PGDAVALGLQCFTLDLKNVT CQWQQQDHAS SQGFFYHSRA RCCPRDRYPI WENCEEEEKTNPGLQTPQFS RCHFKSRNDS IIHILVEVTT APGTVHSYLG SPFWIHQAVRLPTPNLHWRE ISSGHLELEW QHPSSWAAQE TCYQLRYTGE GHQDWKVLEPPLGARGGTLE LRPRSRYRLQ LRARLNGPTY QGPWSSWSDP TRVETATETAWISLVTALHL VLGLNAVLGL LLLRWRSKRS RLLHSDYMNM TPRRPGPTRKHYQPYAPPRD FAAYRSKKVA KKPTNKAPHP KQEPQEINFP DDLPGSNTAAPVQETLHGCQ PVTQEDGKES RISVQERQ SEQ ID NO: 131 vector clone: pIB1110Sequence: MPSWALFMVT SCLLLAPQNL AQVSSEQKLI SEEDLISLVT ALHLVLGLNAVLGLLLLRWR SKRSRLLHSD YMNMTPRRPG PTRKHYQPYA PPRDFAAYRSKKVAKKPTNK APHPKQEPQE INFPDDLPGS NTAAPVQETL HGCQPVTQED GKESRISVQE RQSEQ ID NO: 132 vector clone: pIB1111 Sequence:MPSWALFMVT SCLLLAPQNL AQVSSEQKLI SEEDLQDVSL LASDSEPLKCFSRTFEDLTC FWDEEEAAPS GTYQLLYAYP REKPRACPLS SQSMPHFGTRYVCQFPDQEE VRLFFPLHLW VKNVFLNQTR TQRVLFVDSV GLPAPPSIIKAMGGSQPGEL QISWEEPAPE ISDFLRYELR YGPRDPKNST GPTVIQLIATETCCPALQRP HSASALDQSP CAQPTMPWQD GPKQTSPSRE ASALTAEGGSCLISGLQPGN SYWLQLRSEP DGISLGGSWG SWSLPVTVDL PGDAVALGLQCFTLDLKNVT CQWQQQDHAS SQGFFYHSRA RCCPRDRYPI WENCEEEEKTNPGLQTPQFS RCHFKSRNDS IIHILVEVTT APGTVHSYLG SPFWIHQAVRLPTPNLHWRE ISSGHLELEW QHPSSWAAQE TCYQLRYTGE GHQDWKVLEPPLGARGGTLE LRPRSRYRLQ LRARLNGPTY QGPWSSWSDP TRVETATETAWISLVTALHL VLGLSAVLGL LLLRKRSKRS RLLHSDYMNM TPRRPGPTRKHYQPYAPPRD FAAYRSKKVA KKPTNKAPHP KQEPQEINFP DDLPGSNTAAPVQETLHGCQ PVTQEDGKES RISVQERQ SEQ ID NO: 133 vector clone: pIB1112Sequence: MPSWALFMVT SCLLLAPQNL AQVSSEQKLI SEEDLISLVT ALHLVLGLSAVLGLLLLRKR SKRSRLLHSD YMNMTPRRPG PTRKHYQPYA PPRDFAAYRSKKVAKKPTNK APHPKQEPQE INFPDDLPGS NTAAPVQETL HGCQPVTQED GKESRISVQE RQSEQ ID NO: 134 vector clone: pIB1113 Sequence:MPSWALFMVT SCLLLAPQNL AQVSSEQKLI SEEDLQDVSL LASDSEPLKCFSRTFEDLTC FWDEEEAAPS GTYQLLYAYP REKPRACPLS SQSMPHFGTRYVCQFPDQEE VRLFFPLHLW VKNVFLNQTR TQRVLFVDSV GLPAPPSIIKAMGGSQPGEL QISWEEPAPE ISDFLRYELR YGPRDPKNST GPTVIQLIATETCCPALQRP HSASALDQSP CAQPTMPWQD GPKQTSPSRE ASALTAEGGSCLISGLQPGN SYWLQLRSEP DGISLGGSWG SWSLPVTVDL PGDAVALGLQCFTLDLKNVT CQWQQQDHAS SQGFFYHSRA RCCPRDRYPI WENCEEEEKTNPGLQTPQFS RCHFKSRNDS IIHILVEVTT APGTVHSYLG SPFWIHQAVRLPTPNLHWRE ISSGHLELEW QHPSSWAAQE TCYQLRYTGE GHQDWKVLEPPLGARGGTLE LRPRSRYRLQ LRARLNGPTY QGPWSSWSDP TRVETATETAWISLVTALLL VLGLSAVLGL LLLRWRSKRS RLLHSDYMNM TPRRPGPTRKHYQPYAPPRD FAAYRSKKVA KKPTNKAPHP KQEPQEINFP DDLPGSNTAAPVQETLHGCQ PVTQEDGKES RISVQERQ SEQ ID NO: 135 vector clone: pIB1114Sequence: MPSWALFMVT SCLLLAPQNL AQVSSEQKLI SEEDLISLVT ALLLVLGLSAVLGLLLLRWR SKRSRLLHSD YMNMTPRRPG PTRKHYQPYA PPRDFAAYRSKKVAKKPTNK APHPKQEPQE INFPDDLPGS NTAAPVQETL HGCQPVTQED GKESRISVQE RQSEQ ID NO: 136 vector clone: pIB1115 Sequence:MPSWALFMVT SCLLLAPQNL AQVSSEQKLI SEEDLQDVSL LASDSEPLKCFSRTFEDLTC FWDEEEAAPS GTYQLLYAYP REKPRACPLS SQSMPHFGTRYVCQFPDQEE VRLFFPLHLW VKNVFLNQTR TQRVLFVDSV GLPAPPSIIKAMGGSQPGEL QISWEEPAPE ISDFLRYELR YGPRDPKNST GPTVIQLIATETCCPALQRP HSASALDQSP CAQPTMPWQD GPKQTSPSRE ASALTAEGGSCLISGLQPGN SYWLQLRSEP DGISLGGSWG SWSLPVTVDL PGDAVALGLQCFTLDLKNVT CQWQQQDHAS SQGFFYHSRA RCCPRDRYPI WENCEEEEKTNPGLQTPQFS RCHFKSRNDS IIHILVEVTT APGTVHSYLG SPFWIHQAVRLPTPNLHWRE ISSGHLELEW QHPSSWAAQE TCYQLRYTGE GHQDWKVLEPPLGARGGTLE LRPRSRYRLQ LRARLNGPTY QGPWSSWSDP TRVETATETAWISLVTALHL VLGLNAVLGL LLLRKRSKRS RLLHSDYMNM TPRRPGPTRKHYQPYAPPRD FAAYRSKKVA KKPTNKAPHP KQEPQEINFP DDLPGSNTAAPVQETLHGCQ PVTQEDGKES RISVQERQ SEQ ID NO: 137 vector clone: pIB1116Sequence: MPSWALFMVT SCLLLAPQNL AQVSSEQKLI SEEDLISLVT ALHLVLGLNAVLGLLLLRKR SKRSRLLHSD YMNMTPRRPG PTRKHYQPYA PPRDFAAYRSKKVAKKPTNK APHPKQEPQE INFPDDLPGS NTAAPVQETL HGCQPVTQED GKESRISVQE RQSEQ ID NO: 138 vector clone: pIB1117 Sequence:MPSWALFMVT SCLLLAPQNL AQVSSEQKLI SEEDLQDVSL LASDSEPLKCFSRTFEDLTC FWDEEEAAPS GTYQLLYAYP REKPRACPLS SQSMPHFGTRYVCQFPDQEE VRLFFPLHLW VKNVFLNQTR TQRVLFVDSV GLPAPPSIIKAMGGSQPGEL QISWEEPAPE ISDFLRYELR YGPRDPKNST GPTVIQLIATETCCPALQRP HSASALDQSP CAQPTMPWQD GPKQTSPSRE ASALTAEGGSCLISGLQPGN SYWLQLRSEP DGISLGGSWG SWSLPVTVDL PGDAVALGLQCFTLDLKNVT CQWQQQDHAS SQGFFYHSRA RCCPRDRYPI WENCEEEEKTNPGLQTPQFS RCHFKSRNDS IIHILVEVTT APGTVHSYLG SPFWIHQAVRLPTPNLHWRE ISSGHLELEW QHPSSWAAQE TCYQLRYTGE GHQDWKVLEPPLGARGGTLE LRPRSRYRLQ LRARLNGPTY QGPWSSWSDP TRVETATETAWISLVTALYL VLGLNAVLGL LLLRWRSKRS RLLHSDYMNM TPRRPGPTRKHYQPYAPPRD FAAYRSKKVA KKPTNKAPHP KQEPQEINFP DDLPGSNTAAPVQETLHGCQ PVTQEDGKES RISVQERQ SEQ ID NO: 139 vector clone: pIB1118Sequence: MPSWALFMVT SCLLLAPQNL AQVSSEQKLI SEEDLISLVT ALYLVLGLNAVLGLLLLRWR SKRSRLLHSD YMNMTPRRPG PTRKHYQPYA PPRDFAAYRSKKVAKKPTNK APHPKQEPQE INFPDDLPGS NTAAPVQETL HGCQPVTQED GKESRISVQE RQSEQ ID NO: 140 vector clone: pIB1119 Sequence:MPSWALFMVT SCLLLAPQNL AQVSSEQKLI SEEDLQDVSL LASDSEPLKCFSRTFEDLTC FWDEEEAAPS GTYQLLYAYP REKPRACPLS SQSMPHFGTRYVCQFPDQEE VRLFFPLHLW VKNVFLNQTR TQRVLFVDSV GLPAPPSIIKAMGGSQPGEL QISWEEPAPE ISDFLRYELR YGPRDPKNST GPTVIQLIATETCCPALQRP HSASALDQSP CAQPTMPWQD GPKQTSPSRE ASALTAEGGSCLISGLQPGN SYWLQLRSEP DGISLGGSWG SWSLPVTVDL PGDAVALGLQCFTLDLKNVT CQWQQQDHAS SQGFFYHSRA RCCPRDRYPI WENCEEEEKTNPGLQTPQFS RCHFKSRNDS IIHILVEVTT APGTVHSYLG SPFWIHQAVRLPTPNLHWRE ISSGHLELEW QHPSSWAAQE TCYQLRYTGE GHQDWKVLEPPLGARGGTLE LRPRSRYRLQ LRARLNGPTY QGPWSSWSDP TRVETATETAWISLVTAWCL VLGLSAVLGL LLLRWRSKRS RLLHSDYMNM TPRRPGPTRKHYQPYAPPRD FAAYRSKKVA KKPTNKAPHP KQEPQEINFP DDLPGSNTAAPVQETLHGCQ PVTQEDGKES RISVQERQ SEQ ID NO: 141 vector clone: pIB1120Sequence: MPSWALFMVT SCLLLAPQNL AQVSSEQKLI SEEDLISLVT AWCLVLGLSAVLGLLLLRWR SKRSRLLHSD YMNMTPRRPG PTRKHYQPYA PPRDFAAYRSKKVAKKPTNK APHPKQEPQE INFPDDLPGS NTAAPVQETL HGCQPVTQED GKESRISVQE RQSEQ ID NO: 142 vector clone: pIB1179 Sequence:MALPVTALLL PLALLLHAAR PEPKSCDKTH TCPPCPAPEL LGGPSVFLFPPKPKDTLMIS RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREEQYNSTYRVVS VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPREPQVYTLPPS RDELTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTTPPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLSPGKAAAFWVL VVVGGVLACY SLLVTVAFII FWVRSKRSRL LHSDYMNMTPRRPGPTRKHY QPYAPPRDFA AYRSKKVAKK PTNKAPHPKQ EPQEINFPDDLPGSNTAAPV QETLHGCQPV TQEDGKESRI SVQERQ SEQ ID NO: 143 vector clone:pIB1180 Sequence: MALPVTALLL PLALLLHAAR PAEPKSPDKT HTCPPCPAPP VAGPSVFLFPPKPKDTLMIA RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREEQYNSTYRVVS VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPREPQVYTLPPS RDELTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTTPPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLSPGKKDPKFWV LVVVGGVLAC YSLLVTVAFIIFWVRSKRSR LLHSDYMNMTPRRPGPTRKH YQPYAPPRDF AAYRSKKVAK KPTNKAPHPK QEPQEINFPDDLPGSNTAAP VQETLHGCQP VTQEDGKESR ISVQERQ SEQ ID NO: 144 vector clone:pIB1181 Sequence: MALPVTALLL PLALLLHAAR PERKCCVECP PCPAPPVAGP SVFLFPPKPKDTLMISRTPE VTCVVVDVSH EDPEVQFNWY VDGVEVHNAK TKPREEQFNSTFRVVSVLTV VHQDWLNGKE YKCKVSNKGL PAPIEKTISK TKGQPREPQVYTLPPSREEM TKNQVSLTCL VKGFYPSDIS VEWESNGQPE NNYKTTPPMLDSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPGKFWVLVVVGGVL ACYSLLVTVA FIIFWVRSKR SRLLHSDYMN MTPRRPGPTRKHYQPYAPPR DFAAYRSKKV AKKPTNKAPH PKQEPQEINF PDDLPGSNTAAPVQETLHGC QPVTQEDGKE SRISVQERQ SEQ ID NO: 145 vector clone: pIB1182Sequence: MALPVTALLL PLALLLHAAR PELKTPLGDT THTCPRCPEP KSCDTPPPCPRCPEPKSCDT PPPCPRCPEP KSCDTPPPCP RCPAPELLGG PSVFLFPPKPKDTLMISRTP EVTCVVVDVS HEDPEVQFKW YVDGVEVHNA KTKPREEQYNSTFRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KTKGQPREPQVYTLPPSREE MTKNQVSLTC LVKGFYPSDI AVEWESSGQP ENNYNTTPPMLDSDGSFFLY SKLTVDKSRW QQGNIFSCSV MHEALHNRFT QKSLSLSPGKFWVLVVVGGV LACYSLLVTV AFIIFWVRSK RSRLLHSDYM NMTPRRPGPTRKHYQPYAPP RDFAAYRSKK VAKKPTNKAP HPKQEPQEIN FPDDLPGSNTAAPVQETLHG CQPVTQEDGK ESRISVQERQ SEQ ID NO: 146 vector clone: pIB1183Sequence: MALPVTALLL PLALLLHAAR PESKYGPPCP SCPAPEFLGG PSVFLFPPKPKDTLMISRTP EVTCVVVDVS QEDPEVQFNW YVDGVEVHNA KTKPREEQFNSTYRVVSVLT VLHQDWLNGK EYKCKVSNKG LPSSIEKTIS KAKGQPREPQVYTLPPSQEE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPVLDSDGSFFLY SRLTVDKSRW QEGNVFSCSV MHEALHNHYT QKSLSLSLGKFWVLVVVGGV LACYSLLVTV AFIIFWVRSK RSRLLHSDYM NMTPRRPGPTRKHYQPYAPP RDFAAYRSKK VAKKPTNKAP HPKQEPQEIN FPDDLPGSNTAAPVQETLHG CQPVTQEDGK ESRISVQERQ SEQ ID NO: 147 vector clone: pIB1184Sequence: MALPVTALLL PLALLLHAAR PESKYGPPCP PCPAPEFEGG PSVFLFPPKPKDTLMISRTP EVTCVVVDVS QEDPEVQFNW YVDGVEVHNA KTKPREEQFQSTYRVVSVLT VLHQDWLNGK EYKCKVSNKG LPSSIEKTIS KAKGQPREPQVYTLPPSQEE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPVLDSDGSFFLY SRLTVDKSRW QEGNVFSCSV MHEALHNHYT QKSLSLSLGKFWVLVVVGGV LACYSLLVTV AFIIFWVRSK RSRLLHSDYM NMTPRRPGPTRKHYQPYAPP RDFAAYRSKK VAKKPTNKAP HPKQEPQEIN FPDDLPGSNTAAPVQETLHG CQPVTQEDGK ESRISVQERQ SEQ ID NO: 148 vector clone: pIB1185Sequence: MALPVTALLL PLALLLHAAR PEPKSCDKTH TCPPCPAPEL LGGPSVFLFPPKPKDTLMIS RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREEQYNSTYRVVS VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPREPQVYTLPPS RDELTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTTPPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLSPGKAAAIEVM YPPPYLDNEK SNGTIIHVKG KHLCPSPLFP GPSKPFWVLVVVGGVLACYS LLVTVAFIIF WVRSKRSRLL HSDYMNMTPR RPGPTRKHYQPYAPPRDFAA YRSKKVAKKP TNKAPHPKQE PQEINFPDDL PGSNTAAPVQETLHGCQPVT QEDGKESRIS VQERQ SEQ ID NO: 149 vector clone: pIB1186Sequence: MALPVTALLL PLALLLHAAR PAEPKSPDKT HTCPPCPAPP VAGPSVFLFPPKPKDTLMIA RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREEQYNSTYRVVS VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPREPQVYTLPPS RDELTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTTPPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLSPGKKDPKIEV MYPPPYLDNE KSNGTIIHVK GKHLCPSPLF PGPSKPFWVLVVVGGVLACY SLLVTVAFII FWVRSKRSRL LHSDYMNMTP RRPGPTRKHYQPYAPPRDFA AYRSKKVAKK PTNKAPHPKQ EPQEINFPDD LPGSNTAAPVQETLHGCQPV TQEDGKESRI SVQERQ SEQ ID NO: 150 vector clone: pIB1187Sequence: MALPVTALLL PLALLLHAAR PERKCCVECP PCPAPPVAGP SVFLFPPKPKDTLMISRTPE VTCVVVDVSH EDPEVQFNWY VDGVEVHNAK TKPREEQFNSTFRVVSVLTV VHQDWLNGKE YKCKVSNKGL PAPIEKTISK TKGQPREPQVYTLPPSREEM TKNQVSLTCL VKGFYPSDIS VEWESNGQPE NNYKTTPPMLDSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPGKIEVMYPPPYLD NEKSNGTIIH VKGKHLCPSP LFPGPSKPFW VLVVVGGVLACYSLLVTVAF IIFWVRSKRS RLLHSDYMNM TPRRPGPTRK HYQPYAPPRDFAAYRSKKVA KKPTNKAPHP KQEPQEINFP DDLPGSNTAA PVQETLHGCQPVTQEDGKES RISVQERQ SEQ ID NO: 151 vector clone: pIB1188 Sequence:MALPVTALLL PLALLLHAAR PELKTPLGDT THTCPRCPEP KSCDTPPPCPRCPEPKSCDT PPPCPRCPEP KSCDTPPPCP RCPAPELLGG PSVFLFPPKPKDTLMISRTP EVTCVVVDVS HEDPEVQFKW YVDGVEVHNA KTKPREEQYNSTFRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KTKGQPREPQVYTLPPSREE MTKNQVSLTC LVKGFYPSDI AVEWESSGQP ENNYNTTPPMLDSDGSFFLY SKLTVDKSRW QQGNIFSCSV MHEALHNRFT QKSLSLSPGKIEVMYPPPYL DNEKSNGTII HVKGKHLCPS PLFPGPSKPF WVLVVVGGVLACYSLLVTVA FIIFWVRSKR SRLLHSDYMN MTPRRPGPTR KHYQPYAPPRDFAAYRSKKV AKKPTNKAPH PKQEPQEINF PDDLPGSNTA APVQETLHGCQPVTQEDGKE SRISVQERQ SEQ ID NO: 152 vector clone: pIB1189 Sequence:MALPVTALLL PLALLLHAAR PESKYGPPCP SCPAPEFLGG PSVFLFPPKPKDTLMISRTP EVTCVVVDVS QEDPEVQFNW YVDGVEVHNA KTKPREEQFNSTYRVVSVLT VLHQDWLNGK EYKCKVSNKG LPSSIEKTIS KAKGQPREPQVYTLPPSQEE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPVLDSDGSFFLY SRLTVDKSRW QEGNVFSCSV MHEALHNHYT QKSLSLSLGKIEVMYPPPYL DNEKSNGTII HVKGKHLCPS PLFPGPSKPF WVLVVVGGVLACYSLLVTVA FIIFWVRSKR SRLLHSDYMN MTPRRPGPTR KHYQPYAPPRDFAAYRSKKV AKKPTNKAPH PKQEPQEINF PDDLPGSNTA APVQETLHGCQPVTQEDGKE SRISVQERQ SEQ ID NO: 153 vector clone: pIB1190 Sequence:MALPVTALLL PLALLLHAAR PESKYGPPCP PCPAPEFEGG PSVFLFPPKPKDTLMISRTP EVTCVVVDVS QEDPEVQFNW YVDGVEVHNA KTKPREEQFQSTYRVVSVLT VLHQDWLNGK EYKCKVSNKG LPSSIEKTIS KAKGQPREPQVYTLPPSQEE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPVLDSDGSFFLY SRLTVDKSRW QEGNVFSCSV MHEALHNHYT QKSLSLSLGKIEVMYPPPYL DNEKSNGTII HVKGKHLCPS PLFPGPSKPF WVLVVVGGVLACYSLLVTVA FIIFWVRSKR SRLLHSDYMN MTPRRPGPTR KHYQPYAPPRDFAAYRSKKV AKKPTNKAPH PKQEPQEINF PDDLPGSNTA APVQETLHGCQPVTQEDGKE SRISVQERQ SEQ ID NO: 154 designation CD40 SequenceKKVAKKPTNK APHPKQEPQE INFPDDLPGS NTAAPVQETL HGCQPVTQED GKESRISVQE RQSEQ ID NO: 155 designation CD40_tandem SequenceKKVAKKPTNK APHPKQEPQE INFPDDLPGS NTAAPVQETL HGCQPVTQEDGKESRISVQE RQKKVAKKPT NKAPHPKQEP QEINFPDDLP GSNTAAPVQETLHGCQPVTQ EDGKESRISV QERQ SEQ ID NO: 156 designation CD40_P227ASequence KKVAKKPTNK AAHPKQEPQE INFPDDLPGS NTAAPVQETL HGCQPVTQEDGKESRISVQE RQ

In some embodiments, any one or more of the arrangements below arecontemplated:

-   -   1. An engineered protein that has at least 80%, 85%, 90%, 95%,        96%, 97%, 98%, 99%, 100%, or any integer that is between 80 and        100%, identity to SEQ ID NO: 166, and wherein the sequence is        not SEQ ID NO: 123.    -   2. An engineered protein that has at least 80%, 85%, 90%, 95%,        96%, 97%, 98%, 99%, 100%, or any integer that is between 80 and        100%, identity to SEQ ID NO: 167, and wherein the sequence is        not SEQ ID NO: 123.    -   3. The engineered protein of any one of arrangements 1 or 2,        further comprising a binding domain, CD28 domain, and CD40        domain.    -   4. The engineered protein of any one of arrangements 2-3,        further comprising a signal peptide sequence.    -   5. The engineered protein of arrangement 4, wherein the signal        peptide sequence has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%,        99%, 100%, or any integer that is between 80 and 100%, identity        to the amino acid sequence of SEQ ID NO: 157.    -   6. The engineered protein of any one of arrangements 3-5,        wherein the binding domain comprises a VL sequence, a VH        sequence, and an at least one linker.    -   7. The engineered protein of arrangement 6, wherein the at least        one linker has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%,        100%, or any integer that is between 80 and 100%, identity to        the amino acid sequence of SEQ ID NO: 159 or 161.    -   8. The engineered protein of any one of arrangements 6-7,        wherein the binding domain comprises two linker sequences.    -   9. The engineered protein of arrangement 8, wherein the two        linker sequences have at least 80%, 85%, 90%, 95%, 96%, 97%,        98%, 99%, 100%, or any integer that is between 80 and 100%,        identity to amino acid sequences SEQ ID NO: 159 and SEQ ID NO:        161, respectively.    -   10. The engineered protein of any one of arrangements 6-9,        wherein the VL sequence has at least 80%, 85%, 90%, 95%, 96%,        97%, 98%, 99%, 100%, or any integer that is between 80 and 100%,        identity to the amino acid sequence of SEQ ID NO: 158.    -   11. The engineered protein of any one of arrangements 6-10,        wherein the VH sequence has at least 80%, 85%, 90%, 95%, 96%,        97%, 98%, 99%, 100%, or any integer that is between 80 and 100%,        identity to the amino acid sequence of SEQ ID NO: 160.    -   12. The engineered protein of any one of arrangements 3-11,        wherein the CD40 domain has at least 80%, 85%, 90%, 95%, 96%,        97%, 98%, 99%, 100%, or any integer that is between 80 and 100%,        identity to the amino acid sequence of SEQ ID NO: 165.    -   13. The engineered protein of any one of arrangements 3-12,        wherein the CD28 domain comprises a CD28 transmembrane domain.    -   14. The engineered protein of arrangement 13, wherein the CD28        transmembrane domain has at least 80%, 85%, 90%, 95%, 96%, 97%,        98%, 99%, 100%, or any integer that is between 80 and 100%,        identity to SEQ ID NO: 163.    -   15. The engineered protein of any one of arrangements 3-14,        wherein the CD28 domain comprises a CD28 extracellular domain.    -   16. The engineered protein of arrangement 15, wherein the CD28        extracellular domain has at least 80%, 85%, 90%, 95%, 96%, 97%,        98%, 99%, 100%, or any integer that is between 80 and 100%,        identity to SEQ ID NO: 162.    -   17. The engineered protein of any one of arrangements 3-16,        wherein the CD28 domain comprises a CD28 intracellular domain.    -   18. The engineered protein of arrangement 17, wherein the CD28        intracellular domain has at least 80%, 85%, 90%, 95%, 96%, 97%,        98%, 99%, 100%, or any integer that is between 80 and 100%,        identity to SEQ ID NO: 164.    -   19. The engineered protein of any one of arrangements 1-18,        wherein the protein further comprises 1, 2, 3, 4, 5, or all 6        CDR sequence(s) selected from the group consisting of:

(SEQ ID NO: 168) QASQSLSNLLA,   (SEQ ID NO: 169) GASNLES,(SEQ ID NO: 170) QGGHYSGL, (SEQ ID NO: 171) TNDMN, (SEQ ID NO: 172)VIYSDDTPDYATWAKG, and/or (SEQ ID NO: 173) GHYDSAVYAYALNI.

-   -   20. An engineered protein comprising an amino acid sequence that        is at least 80% identical to the amino acid sequence of SEQ ID        NO: 166 or 167, wherein the amino acid sequence does not include        at least one of: QKLISEEDLE (SEQ ID NO: 174) or

(SEQ ID NO: 175) LVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKI.

-   -   21. The engineered protein of arrangement 20, wherein the        engineered protein lacks both of QKLISEEDLE (SEQ ID NO: 174) and

(SEQ ID NO: 175) LVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKI.

-   -   22. The engineered protein of either of arrangements 20 or 21,        wherein SEQ ID NO: 166 or 167 comprises a sequence that is at        least 80, 90, 95, 96, 97, 98, 99 or 100% identical to SEQ ID NO:        158.    -   23. The engineered protein of either of arrangements 20 or 21,        wherein SEQ ID NO: 166 or 167 comprises a sequence that is at        least 80, 90, 95, 96, 97, 98, 99 or 100% identical to SEQ ID NO:        160.    -   24. The engineered protein of either of arrangements 23, wherein        SEQ ID NO: 166 or 167 comprises a sequence that is at least 80,        90, 95, 96, 97, 98, 99 or 100% identical to SEQ ID NO: 158.    -   25. The engineered protein of any one of arrangements 20-24,        wherein SEQ ID NO: 166 or 167 comprises a sequence that is at        least 80, 90, 95, 96, 97, 98, 99 or 100% identical to SEQ ID NO:        162.    -   26. The engineered protein of any one of arrangements 20-25,        wherein SEQ ID NO: 166 or 167 comprises a sequence that is at        least 80, 90, 95, 96, 97, 98, 99 or 100% identical to SEQ ID NO:        164.    -   27. The engineered protein of any one of arrangements 20-26,        wherein SEQ ID NO: 166 or 167 comprises a sequence that is at        least 80, 90, 95, 96, 97, 98, 99 or 100% identical to SEQ ID NO:        165.    -   28. The engineered protein of any one of arrangements 20-27,        wherein SEQ ID NO: 166 or 167 comprises a sequence that is at        least 80, 90, 95, 96, 97, 98, 99 or 100% identical to SEQ ID NO:        163.    -   29. The engineered protein of any one of arrangements 20-28,        wherein SEQ ID NO: 166 or 167 comprises a sequence that is at        least 80, 90, 95, 96, 97, 98, 99 or 100% identical to SEQ ID NO:        161.    -   30. The engineered protein of any one of arrangements 20-29,        wherein SEQ ID NO: 166 or 167 comprises a sequence that is at        least 80, 90, 95, 96, 97, 98, 99 or 100% identical to SEQ ID NO:        159.    -   31. The engineered protein of any one of arrangements 20-30,        wherein SEQ ID NO: 166 or 167 comprises a sequence that is at        least 80, 90, 95, 96, 97, 98, 99 or 100% identical to SEQ ID NO:        157.    -   32. The engineered protein of any one of arrangements 20-31,        wherein SEQ ID NO: 166 or 167 comprises a sequence that is at        least 80, 90, 95, 96, 97, 98, 99 or 100% identical to SEQ ID NO:        162.    -   33. The engineered protein of any one of arrangements 20-32,        wherein SEQ ID NO: 166 or 167 comprises 1, 2, 3, 4, 5, 6, 7,        and/or all 8 sequence(s) that is/are at least 95% identical to a        sequence selected from the group consisting of SEQ ID NOs: 158,        160, 162, 163, 164, and 165.    -   34. The engineered protein of any one of arrangements 20-33,        wherein SEQ ID NO: 166 or 167 comprises a sequence that is more        than 98% identical to each of SEQ ID NOs: 158, 160, 162, 163,        164, and 165.    -   35. A CoStAR comprising:    -   (a) an optional signal peptide;    -   (b) a binding domain, wherein the binding domain binds to an        anti-pembrolizumab antibody or binding fragment thereof;    -   (c) a CD28 domain;    -   (d) a CD40 domain;    -   wherein a) is optionally linked to b), wherein b) is linked to        c), wherein c) is linked to d), and wherein the CoStAR comprises        an amino acid sequence that:        -   i) lacks at least one of: QKLISEEDLE (SEQ ID NO: 174) or            LVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQV            YSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKI (SEQ ID NO: 175);        -   ii) has an amino acid sequence that is greater than 95%            identical to SEQ ID NO: 166 or 167;        -   iii) has an amino acid sequence that is greater than 80%            identical to SEQ ID NO: 166 or 167 and is not SEQ ID NO:            123; or        -   iv) any combination of i-iv.    -   36. A fusion protein comprising:    -   (a) a means for binding to an antibody that binds to        pembrolizumab;    -   (b) a CD28 domain;    -   (c) a CD40 domain;    -   wherein a) is linked to b), wherein b) is linked to c), and        wherein the fusion protein comprises an amino acid sequence        that:        -   i) lacks at least one of: QKLISEEDLE (SEQ ID NO: 174) or            LVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQV            YSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKI (SEQ ID NO: 175);        -   ii) has an amino acid sequence that is greater than 95%            identical to SEQ ID NO: 166 or 167;        -   iii) has an amino acid sequence that is greater than 80%            identical to SEQ ID NO: 166 or 167 and is not SEQ ID NO:            123; or        -   iv) any combination of i-iv.    -   37. The CoStAR or fusion protein of arrangements 35 or 36,        wherein the binding domain or the means for binding to an        antibody that binds to pembrolizumab comprises: 1, 2, 3, 4, 5,        or all 6 CDR sequence(s) selected from the group consisting of:        QASQSLSNLLA (SEQ ID NO: 168), GASNLES (SEQ ID NO: 169), QGGHYSGL        (SEQ ID NO: 170), TNDMN (SEQ ID NO: 171), VIYSDDTPDYATWAKG (SEQ        ID NO: 172), and/or GHYDSAVYAYALNI (SEQ ID NO: 173).    -   38. The CoStAR or fusion protein of one of arrangements 35-37,        wherein the CD28 domain comprises: SEQ ID Nos: 162, 163, and        164, or a sequence that is at least 80% or 95, or 98% identical        thereto.    -   39. The CoStAR or fusion protein of one of arrangements 35-38,        wherein the CD40 domain comprises: SEQ ID No: 165, or a sequence        that is at least 80% or 95, or 98% identical thereto.    -   40. A fusion protein comprising the amino acid sequence of SEQ        ID NO: 166.    -   41. A fusion protein comprising the amino acid sequence of SEQ        ID NO: 167.    -   42. A nucleic acid which encodes the protein of any one of the        preceding arrangements.    -   43. A vector which comprises the nucleic acid of any one of the        preceding arrangements.    -   44. A cell which expresses the protein of any one of the        preceding arrangements.    -   45. A cell which expresses at least two proteins of any one of        the preceding arrangements.    -   46. A method of making the cell of any one of arrangement 44 or        45, which comprises the step of transducing or transfecting a        cell with a vector of arrangement 43.    -   47. A method for preparing a population of cells that express a        protein of any one of arrangements 1 to 41, comprising detecting        expression of the protein on the surface of cells transfected or        transduced with a vector according to arrangement 43 and        selecting cells which are identified as expressing the protein.    -   48. A cell population produced by the method of arrangement 47.    -   49. A cell population which is enriched for cell expression a        protein of any one of arrangements 1 to 41.    -   50. A method for treating a disease in a subject in need        thereof, which comprises the step of administering the cell of        any one of arrangements 44-45 or the cell population of        arrangement 48 or 49 to the subject.    -   51. The engineered protein of any one of arrangements 1-41,        wherein the binding domain and CD28 domain are connected by at        least one linker.

Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalterations can be made herein without departing from the spirit andscope of the invention as defined in the appended claims.

EXAMPLES

The present invention will be further illustrated in the followingExamples which are given for illustration purposes only and are notintended to limit the invention in any way.

Examples 1

Coculture assay set up. T cells from 2 healthy donors were eithermodified to express the constructs tested or left non-transduced (NTD)at MOI 10. One day prior to coculture set up, effector T cells werethawed and resuspended at 1×10⁶ cells/mL in T cell media (TCM) withoutIL2 and incubated overnight at 37° C. with 5% CO2. On the day ofcoculture, T cells (effectors) and Ba/F3-OKT3 targets were collected andcounted using a ViCELL BLU as per manufacturer's instructions. T cellswere then cocultured with B a/F3 OKT3 targets at the 10:1, 1:1 and 1:10E:T (effector:target) ratios overnight. For the inducible costimulatoryprotein constructs tested (i.e., pIB1097 to pIB1102), an additional setof wells were setup to which 10 μg/mL pembrolizumab was added inaddition to Ba/F3 OKT3 targets. Each condition was performed induplicates. Unstimulated T cells served as negative controls. Two setsof each E:T ratio as well as the T cell only control plates were set up.Brefeldin A was added at 1:1000 dilution to one set of plates to assesscytokine production by intracellular cytokine staining (ICS) using aflow cytometer after overnight co-culture. The second set was incubatedfor 5 days following which T cell counts and activation markerexpression (i.e., 41BB and CD69) was assessed by flow cytometry.

The invention includes modifying components of the TCR complex andassociated signaling adaptors (such as, for example, in a TCRincorporated antigen agnostic receptor “TIAAR”), identifyingtransmembrane domains (TMDs) and modifications that enable constitutiveactivation of receptors (“constitutive”) and utilizing antibodies toinduce activation of the receptor (“inducible”).

scFV targeting co-stimulatory or inhibitor receptors and ligands. ThescFV are derived from antibodies targeting co-stimulatory or inhibitorymolecules expressed on immune cells.

TABLE 9 Signal peptide Tag ECD_TMD ICD Costim GOI description CD8a MychZ270_HL NA CD28-CD40 anti-NKG2A blocking CD8a Myc hZ270_LH NA CD28-CD40anti-NKG2A blocking CD8a Myc Varlilumab_HL NA CD28-CD40 anti-CD27agonist CD8a Myc Varlilumab_LH NA CD28-CD40 anti-CD27 agonist CD8a MycUrelumab_HL NA CD28-CD40 anti-CD137 agonist CD8a Myc Urelumab_LH NACD28-CD40 anti-CD137 agonist CD8a Myc TRX518_HL NA CD28-CD40 anti-GITRagonist CD8a Myc TRX518 LH NA CD28-CD40 anti-GITR agonist CD8a MycPembroluzimab_HL NA CD28-CD40 anti-PD1 blocking CD8a MycPembroluzimab_LH NA CD28-CD40 anti-PD1 blocking CD8a Myc Atezolizumab_HLNA CD28-CD40 anti-PDL1 blocking CD8a Myc Atezolizumab_LH NA CD28-CD40anti-PDL1 blocking CD8a Myc RONK203_HL NA CD28-CD40 anti-FasL blockingCD8a Myc RONK203_LH NA CD28-CD40 anti-FasL blocking CD8a MycTavolimab_HL NA CD28-CD40 anti-OX40 agonist CD8a Myc Tavolimab_LH NACD28-CD40 anti-OX40 agonist CD8a Myc Ipilimumab_HL NA CD28-CD40anti-CTLA4 blocking CD8a Myc Ipilimumab_LH NA CD28-CD40 anti-CTLA4blocking CD8a Myc KY1044_HL NA CD28-CD40 anti-ICOS agonist CD8a MycKY1044_LH NA CD28-CD40 anti-ICOS agonist CD8a Myc APX005_HL NA CD28-CD40anti-CD40 agonist CD8a Myc APX005_LH NA CD28-CD40 anti-CD40 agonist CD8aMyc Selicrelumab_HL NA CD28-CD40 anti-CD40 agonist CD8a MycSelicrelumab_LH NA CD28-CD40 anti-CD40 agonist

TABLE 10 TIAAR (TCR incorporated) list of constructs Signal ECD_ GOICode Concept peptide Tag TMD ICD Costim description pIB1026 TIAAR CD3DMyc CD3D N/A CD28- CD3D_CD3D_ CD40 CD28CD40 pIB1027 TIAAR CD3E FLAG CD3EN/A CD28- CD3E_CD3E_ CD40 CD28CD40 pIB1028 TIAAR CD3G Myc CD3G N/A CD28-CD3G_CD3G_ CD40 CD28CD40 pIB1029 TIAAR CD3Z Myc IC CD3Z N/A CD28-CD3Z_CD3Z_ CD40 CD28CD40_Myc pIB1030 TIAAR CD8A Myc hTRDC N/A CD28-CD8A_hTRDC_ CD40 CD28CD40 pIB1031 TIAAR CD8A FLAG hTRGC1 N/A CD28-CD8A_hTRGC1_ CD40 CD28CD40 pIB1032 TIAAR CD8A Myc mTRAC N/A CD28-CD8A_mTRAC_ CD40 CD28CD40 pIB1033 TIAAR CD8A FLAG mTRBC1 N/A CD28-CD8A_mTRBC1_ CD40 CD28CD40 pIB1046 TIAAR CD8A x2 Myc hTRDC_ N/A CD28-CD8A_hTRDC_ and hTRGC1 CD40 CD28CD40-T2A- FLAG CD8a_hTRGC1_ CD28CD40pIB1047 TIAAR CD8A x2 Myc mTRAC_ N/A CD28- CD8A_mTRAC_ and mTRBC1 CD40CD28CD40-T2A- FLAG CD8A_mTRBC1_ CD28CD40 pIB1048 TIAAR CD3D Myc CD3D_N/A CD28- CD3D_CD3D_ and and CD3E CD40 CD28CD40-T2A- CD3E FLAGCD3E_CD3E_ CD28CD40 pIB1049 TIAAR CD3G Myc CD3G_ N/A CD28- CD3G_CD3G_and and CD3E CD40 CD28CD40-T2A- CD3E FLAG CD3E_CD3E_ CD28CD40 pIB1050TIAAR CD3D Myc CD3D_ CD3D CD28- CD3D_CD3D_ and and CD3E CD3E CD40 CD3DCD3E FLAG (ICD)_ CD28CD40-T2A- CD3E_CD3E_CD3E (ICD)_CD28CD40 pIB1051TIAAR CD3D Myc CD3D_ CD3D N/A CD3D_CD3D_ and and CD3E CD3E CD3D ICD-CD3E FLAG T2A-CD3E_CD3E_ CD3EICD pIB1052 TIAAR CD3D Myc CD3D_ N/A N/ACD3D_CD3D and and CD3E (control)- CD3E FLAG T2A-CD3E_CD3E (control)pIB1053 TIAAR CD3G Myc CD3G_ CD3G_ CD28- CD3G_CD3G_ and and CD3E CD3ECD40 CD3G (ICD)_ CD3E FLAG CD28CD40-T2A- CD3E_CD3E_ CD3E (ICD)_ CD28CD40pIB1054 TIAAR CD3G Myc CD3G_ CD3G_ N/A CD3G_CD3G_ and and CD3E CD3E CD3GICD- CD3E FLAG T2A-CD3E_CD3E_ CD3E ICD pIB1055 TIAAR CD3G Myc CD3G_ N/AN/A CD3G_CD3G and and CD3E (control)- CD3E FLAG T2A-CD3E_ CD3E (control)pIB1056 TIAAR CD3Z Myc IC CD3Z CD3Z CD28- CD3z_CD3z_ CD40 CD3z ICD_CD28CD40_Myc pIB1057 TIAAR CD3Z Myc IC CD3Z CD3Z N/A CD3z_CD3z_ CD3zICD_Myc pIB1058 TIAAR CD3Z Myc IC CD3Z N/A N/A CD3z_CD3z (control)pIB1059 TIAAR CD3Z Myc IC CD3Z CD3Z CD28- CD3Z_CD3Z_CD3Z (x2) CD40 ICD(duplicating CD3z endodomain- 6 ITAMs)_ CD28CD40_Myc pIB1060 TIAAR CD3ZMyc IC CD3Z CD3Z N/A CD3Z_CD3Z_ (x2) CD3Z ICD (duplicating CD3zendodomain-6 ITAMs)_Myc pIB1061 TIAAR CD3Z Myc IC CD3Z CD3Z CD28-CD3Z_CD3Z_ (x2) CD40 CD28CD40_ (swaped) CD3Z (6 ITAMs)_ Myc pIB1062TIAAR CD3Z Myc IC CD3Z CD3Z CD28- CD3Z_CD3Z_ CD40 CD28CD40 (swaped) CD3ZICD_Myc pIB1063 TIAAR CD80 Myc CD80 CD80 N/A CD80 (control) pIB1064TIAAR no Myc IC N/A Lck N/A Lck (control) signal peptide pIB1065 TIAARno Myc IC N/A Lck N/A Lck (Y505F) signal (Y505F) (control) peptidepIB1066 TIAAR CD80 Myc CD80 Lck N/A CD80_Lck pIB1067 TIAAR CD80 Myc CD80Lck CD28- CD80_Lck_ CD40 CD28CD40 pIB1068 TIAAR CD80 Myc CD80 CD80_ N/ACD80_Lck Lck (Y505F) (Y505F) pIB1069 TIAAR CD80 Myc CD80 CD80_ CD28-CD80_Lck Lck CD40 (Y505F)_ (Y505F) CD28CD40 pIB1070 TIAAR CD8A Myc LATLAT N/A LAT (control) pIB1071 TIAAR CD8A Myc LAT LAT CD28- LAT_ CD40C28CD40 pIB1072 TIAAR CD4 Myc CD4 CD4 CD28- CD4 control CD40 pIB1073TIAAR CD4 Myc CD4 CD4 CD28- CD4_CD28_ CD40 CD40 pIB1074 TIAAR CD8A MycCD8A CD8A N/A CD8 control and and and and CD8B FLAG CD8B CD8B pIB1075TIAAR CD8A Myc CD8A CD8A CD28- CD8_CD28_ and and and and CD40 CD40 CD8BFLAG CD8B CD8B

Cytokine production (Bcl-xL, IL2, IFNgamma and TNFalpha) fromgenetically modified and non-transduced T cells (NTD) after overnightstimulation with either Ba/F3 OKT3 targets or left unstimulated (i.e., Tcells only) (FIG. 2 ). There was increased Bcl-xL, IL2, IFNgamma andTNFalpha production from genetically modified T cells as compared to NTDcells in Donor 1. There was an increase in IFNγ production andcomparable or lower levels of Bcl-xL, IL2, IFNgamma and TNFalphaproduction in genetically modified as compared to NTD cells in Donor 2.

Proliferation (T cell counts from CD45+ (TIARR)) and activation markerexpression (41BB from CD45+ and CD69 from CD45+) from geneticallymodified and non-transduced T cells (NTD) after 5-day co-culture witheither Ba/F3 OKT3 targets or left unstimulated (i.e., T cells only)(FIGS. 3A-3B). There was decreased T cell counts in genetically modifiedas compared to NTD cells in Donor 1. There was increased or similar 41BBand CD69 expression in genetically modified as compared to NTD cells inDonor 1. There was increased or similar 41BB and CD69 expression ingenetically modified T cells as compared to NTD cells in Donor 1. Therewas increased T cell counts for 2 modifications tested, and a similar ordecreased T cell counts for the remaining genetically modified T cellsas compared to NTD cells in Donor 2. There was increased or comparable41BB and CD169 expression in genetically modified as compared to NTDcells in Donor 2.

TABLE 11 List of constitutive constructs Signal Code Concept peptide TagECD_TMD ICD Costim GOI description pIB1076 C-SAAR CD8A Myc LZ (cFos)_N/A CD28- LZ (cFos)-EGFRTM/JMD- EGFR CD40 CD28-CD40 pIB1077 C-SAAR CD8AMyc LZ (cFos)_ N/A CD28- LZ (cFos)-CD28TM-CD28- CD28 CD40 CD40 pIB1078C-SAAR CD8A Myc LZ (cJun)_ N/A CD28- LZ (cJun)-EGFRTM/JMD- EGFR CD40CD28-CD40 pIB1079 C-SAAR CD8A Myc LZ (cJun)_ N/A CD28- LZ(cJun)-CD28TM-CD28- CD28 CD40 CD40 pIB1080 C-SAAR CD8A Myc LZ (c/EBP)_N/A CD28- LZ (c/EBP)-EGFRTM/JMD- EGFR CD40 CD28-CD40 pIB1081 C-SAAR CD8AMyc LZ (c/EBP)_ N/A CD28- LZ (c/EBP)-CD28TM- CD28 CD40 CD28-CD40 pIB1103C-SAAR GpA Myc GpA ECD_ N/A CD28- GpA ECD-TMD-CD28-CD40 TMD CD40 pIB1104C-SAAR GpA Myc GpA TMD N/A CD28- GpA TMD-CD28-CD40 CD40 pIB1105 C-SAAREPOR Myc EPOR ECD_ N/A CD28- EpoR ECD-TMD-CD28- TMD CD40 CD40 pIB1106C-SAAR EPOR Myc EPOR TMD N/A CD28- EpoR TMD-CD28-CD40 CD40 pIB1107C-SAAR TPOR Myc TPOR ECD_ N/A CD28- TPO ECD-TPO (WT) TMD - TMD CD40CD28-CD40 pIB1108 C-SAAR TPOR Myc TPOR TMD N/A CD28- TPO (WT) TMD-CD28-CD40 CD40 pIB1109 C-SAAR TPOR Myc TPOR ECD_ N/A CD28- TPO ECD-TPO(S505N) TMD (S505N) CD40 TMD -CD28-CD40 pIB1110 C-SAAR TPOR Myc TPOR TMDN/A CD28- TPO (S505N) TMD -CD28- (S505N) CD40 CD40 pIB1111 C-SAAR TPORMyc TPOR ECD_ N/A CD28- TPO ECD-TPO (W515K) TMD(W515K) CD40TMD-CD28-CD40 pIB1112 C-SAAR TPOR Myc TPOR TMD N/A CD28- TPO (W515K) TMD-CD28- (W515K) CD40 CD40 pIB1113 C-SAAR TPOR Myc TPOR ECD_ N/A CD28- TPOECD-TP (H499L) TMD (H499L) CD40 TMD-CD28-CD40 pIB1114 C-SAAR TPOR MycTPOR TMD N/A CD28- TPO (H499L) TMD-CD28- (H499L) CD40 CD40 pIB1115C-SAAR TPOR Myc TPOR ECD_ N/A CD28- TPO ECD-TPO (S505N- TMD (S505N- CD40W515K) TMD -CD28-CD40 W515K) pIB1116 C-SAAR TPOR Myc TPOR TMD N/A CD28-TPO (S505N-W515K) TMD - (S505N- CD40 CD28-CD40 W515K) pIB1117 C-SAARTPOR Myc TPOR ECD_ N/A CD28- TPO ECD-TPO (H499Y- TMD CD40 S505N)TMD-CD28-CD40 (H499Y- S505N) pIB1118 C-SAAR TPOR Myc TPOR TMD N/A CD28-TPO (H499Y-S505N) TMD - (H499Y- CD40 CD28-CD40 S505N) pIB1119 C-SAARTPOR Myc TPOR ECD_ N/A CD28- TPO ECD-TPO (L498W- TMD CD40 H499C) TMD-CD28-CD40 (L498W- H499C) pIB1120 C-SAAR TPOR Myc TPOR TMD N/A CD28- TPO(L498W-H499C) TMD - (L498W- CD40 CD28-CD40 H499C) pIB1025 C-SAAR CD8aMyc CD28 N/A CD28- CD28 TM_CD28_CD40 CD40 pIB1179 C-SAAR CD8a N/AIgGI28TM N/A CD28- IgG1(CH2CH3)-CD28(TM)- CD40 CD28(CoStim)-CD40(CoStim) pIB1180 C-SAAR CD8a N/A IgG1mut + N/A CD28-IgG1(CH2CH3,mutant)- CIM CD40 CD28(TM)-CD28(CoStim)- CD40(CoStim)pIB1181 C-SAAR CD8al N/A IgG2 + N/A CD28- IgG2(CH2CH3)-CD28(TM)- CD28TMCD40 CD28(CoStim)- CD40(CoStim) pIB1182 C-SAAR I N /A IgG3 + N/A CD28-IgG3(CH2CH3)-CD28(TM)- CD28TM CD40 CD28(CoStim)- CD40(CoStim) pIB1183C-SICD8a CD8a N/A IgG4 + N/A CD28- IgG4(CH2CH3)-CD28(TM)- CD28TM CD40CD28(CoStim)- CD40(CoStim) pIB1184 C-SAAR CDI/A N/A IgG4mut + N/A CD28-IgG4(CH2CH3,mutant)- CD28TM CD40 CD28I-CD28(CoStim)- CD40(CoStim)pIB1185 C-SAAR CD8a N/A IgG1 + CD28 N/A CD28- IgG1(CH2CH3)- stalk/TMCD40 CD28(Stalk + TM)- CD28(CoStim)- CD40(CoStim) pIB1186 C-SAAR CD8aN/A IgG1mut + N/A CD28- IgG1(CH2CH3,mutant)- CD28 stalk/ CD40CD28(Stalk + TM)- TM CD28(CoStim)- CD40(CoStim) pIB1187 C-SAAR CD8a N/AIgG2 + CD28 N/A CD28- IgG2(CH2CH3)- stalk/TM CD40 CD28(Stalk + TM)-CD28(CoStim)- CD40(CoStim) pIB1188 C-SAAR CD8a N/A IgG3 + CD28 N/A CD28-IgG3(CH2CH3)- stalk/TM CD40 CD28(Stalk + TM)- CD28(CoStim)- CD40(CoStim)pIB1189 C-SAAR CD8a N/A IgG4 + CD28 N/A CD28- IgG4(CH2CH3)- stalk/TMCD40 CD28(Stalk + TM)- CD28(CoStim)- CD40(CoStim) pIB1190 C-SAAR CD8aN/A IgG4mut + N/A CD28- IgG4(CH2CH3,mutant)- CD28 stalk/ CD40CD28(Stalk + TM)- TM CD28(CoStim)- CD40(CoStim)

Cytokine production (Bcl-xL, IL2, IFNgamma and TNFalpha) fromgenetically modified and non-transduced T cells (NTD) after overnightstimulation with either Ba/F3 OKT3 targets or left unstimulated (i.e., Tcells only) (FIG. 4 ). There was increased or comparable Bcl-xL, IL2,and TNFalpha production and comparable or lower levels of IFNgamma fromgenetically modified T cells as compared to NTD cells in Donor 1. Therewas an increase in IFNgamma and IL2 production and comparable or lowerlevels of Bcl-xL and TNFalpha production in genetically modified ascompared to NTD cells in Donor 2.

Proliferation (T cell counts from CD45+(LZ)) and activation markerexpression (41BB from CD45+ and CD69 from CD45+) from geneticallymodified and non-transduced T cells (NTD) after 5-day co-culture witheither Ba/F3 OKT3 targets or left unstimulated (i.e., T cells only)(FIGS. 5A-5B). There was decreased T cell counts in genetically modifiedas compared to NTD cells in Donor 1. There was increased 41BB and CD69expression in genetically modified T cells as compared to NTD cells inDonor 1. There was increased T cell counts as compared to NTD cells inDonor 2, an increase in 41BB expression in genetically modified T cellsas compared to NTD cells, and similar or decreased expression of CD169in genetically modified T cells as compared to NTD cells in Donor 2.

TABLE 12 List of inducible constructs Signal Code Concept peptide TagECD_TMD ICD Costim GOI description pIB1082 Inducible EGFR Myc EGFR N/ACD28- WT EGFR ECD CD40 EGFRTM/JMD-CD28-CD40 pIB1083 Inducible EGFR MycEGFR N/A CD28- domain IV - EGFRTM/JMD- (domain IV) CD40 CD28-CD40pIB1084 Inducible EGFR Myc EGFR (623- N/A CD28- EGFRTM/JMD-CD28-CD40668) CD40 (control) pIB1085 Inducible Her2 Myc Her2 N/A CD28- Her 2(Domain 1 to IV)- CD40 TMD/JMD-CD28-CD40 pIB1086 Inducible Her2 Myc Her2N/A CD28- Her 2 (Domain 1 to IV)-TMD (V659E) CD40 (V659E)/JMD-CD28-CD40pIB1087 Inducible Her2 Myc Her2 N/A CD28- Her 2 (Domain 1 to IV)-TMD(V660D) CD40 (G660D)/JMD-CD28-CD40 pIB1088 Inducible Her2 Myc Her2 N/ACD28- Her 2 (Domain 1 to IV)-TMD (V660R) CD40 (G660R)/JMD-CD28-CD40pIB1089 Inducible Her2 Myc Her2 domain N/A CD28- Her 2 (DomainIV)-TMD/JMD- IV_TMD CD40 CD28-CD40 pIB1090 Inducible Her2 Myc Her2domain N/A CD28- Her 2 (Domain IV)-TMD IV_TMD CD40 (V659E)/JMD-CD28-CD40(V659E) pIB1091 Inducible Her2 Myc Her2 domain N/A CD28- Her 2 (DomainIV)-TMD IV_TMD CD40 (G660D)/JMD-CD28-CD40 (G660D) pIB1092 Inducible Her2Myc IV_TMD N/A CD28- Her 2 (Domain IV)-TMD Her2 domain CD40(G660R)/JMD-CD28-CD40 (G660R) pIB1093 Inducible Her2 Myc Her2 TMD N/ACD28- Her 2 TMD/JMD-CD28-CD40 CD40 pIB1094 Inducible Her2 Myc Her2 TMDN/A CD28- Her 2 TMD (V659E)/JMD- (V659E) CD40 CD28-CD40 pIB1095Inducible Her2 Myc Her2 TMD N/A CD28- Her 2 TMD (G660D)/JMD- (G660D)CD40 CD28-CD40 pIB1096 Inducible Her2 Myc Her2 TMD N/A CD28- Her 2 TMD(G660R)/JMD- (G660R) CD40 CD28-CD40 pIB1097 Inducible CD8A Myc A30514N/A CD28- Anti-ID1 VH-VL (A30514- VH_VL CD40 pembrolizumab)-CD28TMDCD28-CD40 pIB1098 Inducible CD8A Myc A30514 N/A CD28- Anti-ID1 VL-VH(A30514- VL_VH CD40 pembrolizumab)-CD28TMD CD28-CD40 pIB1099 InducibleCD8A Myc A30523 N/A CD28- Anti-ID2 Vh-VL (A30523- VH_VL CD40pembrolizumab)-CD28TMD CD28-CD40 pIB1100 Inducible CD8A Myc A30523 N/ACD28- Anti-ID2 VL-Vh (A30523- VL_VH CD40 pembrolizumab)-CD28TMDCD28-CD40 pIB1101 Inducible CD8A Myc A30633 N/A CD28- Anti-ID3 Vh-VL(A30633- VH_VL CD40 pembrolizumab)-CD28TMD CD28-CD40 pIB1102 InducibleCD8A Myc A30633 N/A CD28- Anti-ID3 VL-VH (A30633- VL_VH CD40pembrolizumab)-CD28TMD CD28-CD40

Cytokine production (Bcl-xL, IL2, IFNg and TNFa) from geneticallymodified and non-transduced T cells (NTD) after overnight stimulationwith either Ba/F3 OKT3 targets or Ba/F3 OKT3 targets with 10 ug/mLpembrolizumab or left unstimulated (i.e., T cells only) (FIG. 6 ). Therewas increased or comparable Bcl-xL, IL2, IFNgamma and TNFalphaproduction from genetically modified T cells in the presence of Ba/F3OKT3 and pembrolizumab as compared to conditions with Ba/F3 OKT3stimulation alone and NTD cells in both Donor 1 and Donor 2.

Proliferation (T cell counts from CD45+ (Inducible)) and activationmarker expression (41BB from CD45+ and CD69 from CD45+) from geneticallymodified and non-transduced T cells (NTD) after 5-day co-culture witheither Ba/F3 OKT3 targets or Ba/F3 OKT3 targets with 10 ug/mLpembrolizumab or left unstimulated (i.e., T cells only) (FIGS. 7A-7B).There was increased, comparable and decreased T cell counts ingenetically modified T cells as compared to NTD cells in Donor 1. Therewas increased or similar 41BB and CD169 expression in geneticallymodified T cells as compared to NTD cells in Donor 1. There wasincreased or comparable T cell counts in genetically modified T cells ascompared to NTD cells in Donor 2. There was increased, similar anddecreased 41BB and CD169 expression in genetically modified cells ascompared to NTD cells in Donor 2. Increased T cell counts and activationmarker expression in both donors were observed when the geneticallymodified T cells were stimulated with Ba/F3 OKT3 in the presence ofpembrolizumab compared to conditions with Ba/F3 OKT3 stimulation aloneas well as NTD cells.

Example 2 Use of Sequences in a Cell System for the Treatment of aPatient

An engineered protein having the sequence of SEQ ID NO: 166 will betransfected into a Tumor Infiltrating Lymphocyte (TIL) cell usingstandard procedures by incorporating vectors. This cell will then beused to generate a population of TIL cells expressing those proteins.The population will be derived through detecting expression of theprotein on the surface of the cells transfected to express the twoproteins, and selecting cells which are identified as expressing thoseproteins. Through this process, the population of cells will be enrichedfor those expressing the protein. Following enrichment, the TIL cellswill be administered at a therapeutic amount to a patient as atherapeutic treatment for cancer.

Example 3

Transduction of CoStAR Constructs into TIL Cells

CoStAR constructs were transduced into TIL cells from tumor digests. Theefficiency of CEA or FOLR expression on tumor digests at Day 1 is asshown in FIGS. 20-22 , and in Table 13.

TABLE 13 % CEA from % FOLR1 % CEA+ % CD45- expression FOLR+ Construct(FSC-H (FSC-H (CEA Tumor type Name vs CEA) vs FOLR1) vs FOLR1) CRC CRC11959 71.7 4.53 2.65 (metastatic) CRC CRC 11974 54.6 9.9 5.31(metastatic) NSCLC NSCLC- 3.88 15.3 1.56 9332 NSCLC NSCLC- 15.1 34.814.7 9596 Ovarian OV-9662 2.03 41.3 0.93 Melanoma MEL-CC50 1.37 0.770.061 Melanoma MEL- 1.26 4.53 0.25 11909 Melanoma MEL- 4.4 1.41 0.1817614

The efficiency of CEA or FOLR expression by Day 21 is as shown in FIGS.12-14 . As can be shown in FIGS. 12-14 and 20-22 , the amount of CEA andFOLR expression was significantly higher by 21 days of treatmentcompared to 1 day of treatment. The transduction was performed using thefollowing materials and methodology:

Methodology: Experimental Outline

On day 1, the tumor digest was thawed (no stimulation) in media 1. Therewere a total of 15 tumor digests, and comprised pancreatic, CRC, NSCLC,ovarian, melanoma, and cervical tumors (FIG. 18 ). 0.5-1e6 cells wereseeded per condition (6 conditions). Phenotype was recorded wereapplicable. On days 3 and 4, cells were transduced in media 1 at MOI 10.On day 8, cells were given outgrowth feed, through the addition of 1 mLof media 2 with gentamycin/amphotericin and fresh IL2 (6000 U/mL). Onday 10, static REP (G-REX 6) was performed; cells were stimulated withOKT3 (30 ng/mL)+IL2 (3000 U/mL)+irradiated feeders (1:200). On day 15,dynamic REP was performed; cells were transferred to G-REX 6M with 100mL media 3. On day 18, cells were counted, split as needed, and/orremoved 65 mL media and replenished with fresh media 3. On day 21, theREP was ended. This timeline is also schematically depicted in FIG. 19 .

Methodology: Media Preparation

TCM base media: GMP/TCM media+25 mM HEPES+25 μM 2-Mercaptoethanol

Media 1: TCM base media+10% FBS, 1× Gentamycin/Amphotericin (500×stock)+50 ug/mL vancomycin+3000 IU/mL IL2

Media 2: TCM base media+10% FBS, 2× Gentamicin/Amphotericin B (500×stock)+100 ug/mL vancomycin+6000 IU/mL IL2

Media 3: TCM base media+8% human AB serum, 3000 IU/mL IL2

Methodology: Detailed Protocol by Day

Day 1: Thaw frozen tumor digest

-   -   1. Prepare media 1 as per table in excel sheet. Add 200 uL IL2        to 200 mL of media 1    -   2. Label a 15 ML conical tube for each tumor digest vial    -   3. Remove the tumor digest vials from LN, and immediately thaw        in 37-degree water bath until small chunks remain.    -   4. Sterilize the exterior and bring the vials to BSC.    -   5. Add in 1 ml of pre-warmed media 1 very gently, drop by drop,        to each vial.    -   6. Transfer the total of 2 ml cell suspension from the vial        using a 5 ml serological pipette, and add very gently, drop by        drop, to the match labeled 15 ml conical tube which contains 2        ml of media 1.    -   7. Add in 9 ml of pre-warmed media 1 very gently to the cell        suspension in the 15 ml conical tube.    -   8. Centrifuge at 400 g for 10 min.    -   9. Aspirate off the supernatant carefully without touching the        cell pellet.    -   10. Based on the frozen cell number, resuspend the cells in 5 ml        of media 1 gently    -   11. Count using Vicell and record in excel sheet (1:10 dilution;        30 uL cell suspension in 270 uL media 1)    -   12. Add 1 mL media and split into 6 conditions    -   13. Resuspend cells in either 0.5 M/mL    -   14. Seed 0.5 M/mL in a 24 well plate or 1 M/2 mL in 6 well plate    -   15. Seed 6 wells for each tumor digest (pIB1322, pIB1324,        CTP386.1, CTP387.1, CTP025 and NTD T cells)    -   16. Add 10 uL/mL amphotericin B only for CRC samples    -   17. Place plates in incubator    -   18. Collect cells if available for checking for CEA and FOLR        expression on tumor

Day 3: TIL Transduction During Outgrowth

-   -   1. Prepare required media 1 and add 1 uL/mL IL2 (25 uL IL2 to 25        mL media 1)    -   2. Dilute the needed volume of LVV in 0.1 mL for 24 well plate        or 0.5 ml for 6 well plate with Media 1 in a separate vial for        each sample, gently pipette to mix well and add into cell        culture.    -   3. Add equivalent volume of media 1 alone to the nontransduced        well.    -   4. Mix gently and place plate in the incubator.

Day 4: TIL Second Transduction

-   -   1. Prepare required media 1 and add 1 uL/mL IL2 (35 uL IL2 to 35        mL media 1)    -   2. Use the TVC and LVV volume calculation from day 3, dilute the        needed volume of LVV in 0.1 ml with Media 1 in a separate vial        for each sample    -   3. Mix gently and place plate in the incubator.

Day 8: Outgrowth Feed

-   -   1. Prepare media 2 as per table in the excel sheet. Add 200 uL        IL2 to 100 mL media 2.    -   2. Mix each well    -   3. Add 1 ml of fresh Media 2 to each well for 6 well plate and        500 uL for 24 well plate    -   4. Continue the culture to day 10

Day 10: Static REP

-   -   1. Prepare media 3 as per table in the excel sheet    -   2. Collect all conditions    -   3. Count using Vicell and record in excel sheet    -   4. Calculate required number of irPBMC feeders needed        -   a. Assuming 0.1e6 cells per condition based on cell counts            below        -   b. At 1:200—will need 12e9 irradiated feeders total        -   c. Thaw feeders in media 3 and centrifuge at 300 g×5 mins        -   d. Count feeders using Vicell    -   5. Prepare master mix of media 3+OKT3+IL2 as needed, using the        ratios outline in Table 14 below.

TABLE 14 Stock Per mL of Volume Reagent concentration media 4 neededMedia 3 NA 1 mL 30 mL OKT3 (30 ng/mL 100 ug/mL NA 30 uL (100 ng/ml) or0.3 uL/mL) IL2 3000 U/uL  1 uL  30 uL 

-   -   6. Add required volumes in the following order to each well of a        6-well G-Rex        -   a. Add 30 mL media 4+OKT3+IL2 first to each well        -   b. Add irradiated feeders to the bottom of the well        -   c. Add TILs gently to the bottom of well    -   7. Plate an extra well of irradiated feeders alone to monitor        and phenotype (seeded in Grex as well)

Day 15: Dynamic REP

-   -   1. Collect all cell culture, resuspend wells    -   2. Count using Vicell and record in excel sheet    -   3. Transfer 30 mL cell suspension to 6M G-REX and top to 100 ml        (ie, add 60 mL media 3+10 mL to rinse the well)    -   4. Add fresh IL2 (1 uL/mL for 3000 U/mL IL2). Add 100 uL IL2 for        each condition

Day 18: Dynamic REP Maintenance

-   -   1. Take 6M G-REX from incubator    -   2. Resuspend cells in the wells    -   3. Count using Vicell and record in excel sheet    -   4. Either do a media change for cell counts<1e6 cells/mL or        split conditions with cell counts>1e6 cells/mL    -   Notes:        -   a. Let G-Rex sit in Biosafety cabinet for 30 minutes if            cells are in suspension or disturbed prior to removing            media.        -   b. For media change, remove 60 mL media and replenish with            60 mL media 4+IL2        -   c. For splitting conditions, transfer 50 mL cell suspension            to a second G-REX 6M and make up volume to 100 mL for all            conditions.    -   5. Top wells with media 4 to 100 ml and place in incubator.

Day 21: Harvest

-   -   1. Harvest all cells.    -   2. Centrifuge at 400 g×10 mins    -   3. Resuspend in 50 mL media 3 and pass through 100 um cell        strainer into a fresh 50 mL conical tube    -   4. Count using Vicell and record in excel sheet    -   5. Spin down cells at 400 g for 10 min and aspirate off the        supernatant.    -   6. Cryopreserve the cells with CryoStor CS10 and freeze in        cryovials.

Example 4 TIL Functional Screening

TIL cells expressing CoStAR constructs then underwent a screen to assessfunction. The results of this screen are as shown in FIGS. 17A-17B. Fromthe screening, a population of cells were generated that were enrichedfor CEA and/or FOLR expression. The fraction of anti-CEA, anti-FOLR, anduniversal CoStAR cells with positive expression are as shown in FIGS.23-27 . Of these TILS, the CD4/8 ratio was as shown in FIGS. 28A-28D.The methodology for the assay was as follows:

Methodology: Experiment Outline

On day 1, TIL cells were thawed. On day 2, CoStAR-modified TILS weresorted, then ran on fortessa. On day 4, the media change was completed,and the cells were stained and ran on fortessa. On day 6, the TCM mediawas changed to exclude IL2. On day 7, the co-culture and serialstimulation assay was set up. As can be seen in FIGS. 30A-30H, 31A-31H,and 32A-32H, the co-culture with autologous digest is inconclusiveprobably due to variability introduced in tumor reactivity of the TILsbetween different conditions as a result of sorting for CoStAR.Additionally, a higher level of IL2 production observed from bothanti-CEA and Universal CoStAR modified TILs in the presence of signal1+2 in 24-hour co-culture assay. Similar trends observed for IFNg andTNFa. On day 8, the supernatant was collected, and MSD was performed.Cells were then stained to assess the expression of activation markers.An outline of this time is as shown in FIG. 29 .

Methodology: TCM Media with IL2

To a bottle of 500 mL RPMI 1640, add: 50 mL FBS, 5 mL pen/strep, 5 mLHEPES, 500 uL 2-mercaptoethanol, and 1 uL/mL of IL-2 stock (3e6 U/mL).The final concentration of the media should be 3000 U/mL.

Methodology: Detailed Protocol by Day

Day 1: Thaw TILS

-   -   1) Place vials in water bath    -   2) Transfer to hood and add cell suspension to 9 mL TCM media    -   3) Centrifuge at 400 g×5 mins    -   4) Aspirate    -   5) Add 5 mL media    -   6) Count using Vicell    -   7) Rest cells overnight at 1e6 cells/mL

Day 2: Sort CoStAR-modified TILS

-   -   1. Collect cells    -   2. Centrifuge at 400 g×5 mins    -   3. Aspirate and resuspend in 5 mL media    -   4. Count using Vicell    -   5. Prepare buffer (10% FBS+2 mM EDTA), add following to 500 mL        of RPMI 1640        -   50 mL of fetal bovine serum        -   2 mL of 0.5M EDTA    -   6. Determine cell number, collect cells for LS columns (see        “Pre- and Post-sort counts”).    -   7. Centrifuge@400×g for 5 minutes, aspirate supernatant. Break        pellet.    -   8. Transfer to 15 mL conical tubes    -   9. Incubate cells with antibodies as per table        -   For CTP205, add 1 uL sol FOLR Fc APC antibody per 1e6 cells        -   For pIB1322 and pIB1324, add 10 uL donkey anti-rabbit PE            antibody per 1e6 cells        -   For CTP386.1 and 387.1, add 2.5 uL CEACAM5-Fc FITC antibody            per 1e6 cells    -   10. Mix well, incubate in the dark@4 C for 30 minutes.    -   11. Wash cells by adding 1-2 mL buffer per 10e6 cells    -   12. Centrifuge@400×g for 5 minutes. Aspirate supernatant.    -   13. Add 5 mL buffer to wash.    -   14. Centrifuge@400×g for 5 minutes. Aspirate supernatant.    -   15. Resuspend in 80 uL of buffer 10e6 cells (see table)    -   16. Add 20 uL of microbeads 10e6 cells (see table)    -   17. Mix well, incubate for 15 minutes@4 C    -   18. Wash cells by adding 1-2 mL buffer per 10e6 cells    -   19. Centrifuge@400×g for 5 minutes. Aspirate supernatant.    -   20. Resuspend up to 100e6 cells in 500 uL of buffer.    -   21. Magnetic separation using LS column        -   a) Place LS column on MACS separator.        -   b) Rinse with 2 mL buffer. Use column immediately. Rinse            column right before adding cell suspension.        -   c) Apply cell suspension to column        -   d) Wash column 3× with 2 mL of buffer. Collect unlabelled            cells that pass through the column        -   e) Remove column form separator and place on appropriate            tube.        -   f) Pipette 5 mL buffer onto column.        -   g) Immediately flush by firmly pushing plunger into column.        -   h) Centrifuge at 400 g×5 mins        -   i) Resuspend in 1 mL (or 5 mL, depending on pellet) TCM            media+3000 U/mL IL2        -   j) Count on Vicell. (see Pre- and post-sort counts)        -   k) Add media to maintain cells at 1e6/mL and incubate flasks

Day 4: Complete Media Change

-   -   1. Collect cells in tubes    -   2. Centrifuge at 400 g×5 mins    -   3. Aspirate    -   4. Add TCM media with IL2    -   5. Count using Vicell    -   6. Add media to maintain cells at 1e6 cells/mL    -   7. Collect 0.05 to 0.1e6 cells to run on fortessa-stain with L/D        Near Far IR

Day 6: Change to Media without IL2

-   -   8. Collect cells in tubes    -   9. Centrifuge at 400 g×5 mins    -   10. Aspirate    -   11. Add TCM media WITHOUT IL2    -   12. Count using Vicell    -   13. Add TCM media WITHOUT IL2 media to maintain cells at 1e6        cells/mL

Day 7: Set Up Co-Culture

-   -   1. Collect sorted TILs    -   2. Centrifuge at 400 g×5 mins    -   3. Aspirate and add 5 mL TCM media without IL2    -   4. Count using Vicell    -   5. Collect targets—K562 WT, OKT3, CEACAM5 and OKT3 CEACAM5 and        OKT3 FOLR    -   6. Thaw autologous digest in TCM media without IL2    -   7. Centrifuge at 400 g×5 mins    -   8. Aspirate and resuspend in 5 ml TCM media without IL2    -   9. Count effectors, target cell lines and autologous digest        using Vicell    -   10. Collect 2e6 cells and resuspend in 2 mLs final volume with        TCM media without IL2    -   11. Collect 1e6 targets and resuspend in 10 mLs final volume        with TCM media without IL2    -   12. Collect either 2e6 or 10e6 cells for autologous digest and        resuspend in 2 mL TCM media without IL2 (this will be either 1:1        or 1:5 ratio)    -   13. Plate 50 uL TILs as per plate map    -   14. Plate 50 uL targets as per plate map.    -   15. Resuspend targets according to TILs to adjust E:T ratio to        10:1 (this is only for cell lines)    -   16. Prepare pembro from stock (5 mg/mL) as per table    -   17. Add 100 uL pembro as per plate map    -   18. Add 100 uL media to plate 1    -   19. Make up volume to 200 uL final in all wells. Note that        effectors only and target only wells will have 150 uL; add 50 uL        media or appropriate pembro concentration depending on plates    -   20. Add TILs for FMOs    -   21. Add PBS to empty wells    -   22. Plate plates in the incubator

Example 5 Serial Stimulation Assay of CoStAR-TIL Cells

TIL cells expressing CoStAR constructs then underwent a serialstimulation assay. The results of the stimulation assay are as shown inFIGS. 15A-15E and 16A-16E. As can be seen in those figures, the TILsmodified with anti-CEA CoStAR but not Universal CoStAR expanded in aserial stimulation assay comparable to anti-FOLR CoStAR modified TILs.The methodology for the stimulation assay was as follows:

Day 1:

-   -   1. Collect effector cells and count using Vicell    -   2. Thaw targets (irradiated K562 OKT3 CEACAM5 and K562 OKT3        FOLR1) and count using Vicell    -   3. Record counts in excel sheet and make up volume to 0.2M/ml        for each sample    -   4. Plate 50 uL of effectors (ie, 50K cells) and targets (ie, 10K        cells) as per plate map. Note: ADD K562 OKT3 FOLR to control        plate    -   5. Add 100 ul of media into appropriate wells as per plate map    -   6. Dilute 10 uL of stock pembro in 4.99 ml of fresh media to        make 2× concentration for plate 2 (10 ug/ml) and add 100 ul into        appropriate wells as per plate map    -   7. Dilute 0.5 mL of stock pembro in 4.5 ml of fresh media to        make 2× concentration for plate 3 (500 ug/ml) and add 100 ul        into appropriate wells as per plate map    -   8. Add 200 uL PBS to wells at the edge of the plates    -   9. Place the plates into incubator

Day 6, 13, 20, 27, and 34: Collect cells for cell count

-   -   1. Prepare cell counting by adding 180 ul of media into the        Vi-cell 96 well counting plates    -   2. Resuspend cells in the coculture plates    -   3. Take 20 ul of resuspended cells and add into the Vi-cell 96        well plates. Refer to “plate map for counting” tab in the excel        sheet.    -   4. Put the coculture plates back into the incubator    -   5. Count the Vi-cell 96 well plates (1:10 dilution)    -   6. Put plates back in the incubator

Day 7, 14, 21, 28, and 35: re-stimulation of cells with targets

-   -   1. Take the cell count files from vi-cell and plug into the        excel sheet for calculation    -   2. Collect 50 uL sample for staining    -   3. Seal plates with parafilm and spin down the coculture plates        at 500 g×5 min    -   4. Decant the plates inside the biosafety cabinet    -   5. Resuspend the cells in 50 ul/well of fresh media    -   6. Prepare pembro and add 100 ul of pembro based on        concentration calculation    -   7. Dilute 12 uL of stock pembro in 6 ml of fresh media to make        2×concentration (10 ug/ml) and add 100 ul into appropriate wells        as per plate map    -   8. Dilute 0.6 mL of stock pembro in 5.4 ml of fresh media to        make 2×concentration (500 ug/ml) and add 100 ul into appropriate        wells as per plate map    -   9. Add 100 ul of media into appropriate wells as per plate map    -   10. Add additional media based on calculation in table    -   11. Thaw irradiated K562 OKT3 CEACAM5 and K562 OKT3 FOLR targets    -   12. resuspend target cells at 1M/ml and add target cells based        on calculation in table    -   13. Mix wells and place in incubator    -   14. Add PBS to empty wells along the edges

Having thus described in detail preferred embodiments of the presentinvention, it is to be understood that the invention defined by theabove paragraphs is not to be limited to particular details set forth inthe above description as many apparent variations thereof are possiblewithout departing from the spirit or scope of the present invention.

What is claimed is:
 1. An engineered protein that has at least 80%, 85%,90%, 95%, 96%, 97%, 98%, 99%, 100%, or any integer that is between 80and 100%, identity to SEQ ID NO: 166, and wherein the sequence is notSEQ ID NO:
 123. 2. An engineered protein that has at least 80%, 85%,90%, 95%, 96%, 97%, 98%, 99%, 100%, or any integer that is between 80and 100%, identity to SEQ ID NO: 167, and wherein the sequence is notSEQ ID NO:
 123. 3. The engineered protein of any one of claim 1 or 2,further comprising a binding domain, CD28 domain, and CD40 domain. 4.The engineered protein of any one of claims 2-3, further comprising asignal peptide sequence.
 5. The engineered protein of claim 4, whereinthe signal peptide sequence has at least 80%, 85%, 90%, 95%, 96%, 97%,98%, 99%, 100%, or any integer that is between 80 and 100%, identity tothe amino acid sequence of SEQ ID NO:
 157. 6. The engineered protein ofany one of claims 3-5, wherein the binding domain comprises a VLsequence, a VH sequence, and an at least one linker.
 7. The engineeredprotein of claim 6, wherein the at least one linker has at least 80%,85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or any integer that is between80 and 100%, identity to the amino acid sequence of SEQ ID NO: 159 or161.
 8. The engineered protein of any one of claims 6-7, wherein the VLsequence has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, orany integer that is between 80 and 100%, identity to the amino acidsequence of SEQ ID NO:
 158. 9. The engineered protein of any one ofclaims 6-8, wherein the VH sequence has at least 80%, 85%, 90%, 95%,96%, 97%, 98%, 99%, 100%, or any integer that is between 80 and 100%,identity to the amino acid sequence of SEQ ID NO:
 160. 10. Theengineered protein of any one of claims 3-9, wherein the CD40 domain hasat least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or any integerthat is between 80 and 100%, identity to the amino acid sequence of SEQID NO:
 165. 11. The engineered protein of any one of claims 3-10,wherein the CD28 domain comprises a CD28 transmembrane domain.
 12. Theengineered protein of claim 11, wherein the CD28 transmembrane domainhas at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or anyinteger that is between 80 and 100%, identity to SEQ ID NO:
 163. 13. Theengineered protein of any one of claims 3-12, wherein the CD28 domaincomprises a CD28 extracellular domain.
 14. The engineered protein ofclaim 13, wherein the CD28 extracellular domain has at least 80%, 85%,90%, 95%, 96%, 97%, 98%, 99%, 100%, or any integer that is between 80and 100%, identity to SEQ ID NO:
 162. 15. The engineered protein of anyone of claims 3-14, wherein the CD28 domain comprises a CD28intracellular domain.
 16. The engineered protein of claim 15, whereinthe CD28 intracellular domain has at least 80%, 85%, 90%, 95%, 96%, 97%,98%, 99%, 100%, or any integer that is between 80 and 100%, identity toSEQ ID NO:
 164. 17. The engineered protein of any one of claims 1-16,wherein the protein further comprises 1, 2, 3, 4, 5, or all 6 CDRsequence(s) selected from the group consisting of: QASQSLSNLLA (SEQ IDNO: 168), GASNLES (SEQ ID NO: 169), QGGHYSGL (SEQ ID NO: 170), TNDMN(SEQ ID NO: 171), VIYSDDTPDYATWAKG (SEQ ID NO: 172), and/orGHYDASVYAYALNI (SEQ ID NO: 173).
 18. An engineered protein comprising anamino acid sequence that is at least 80% identical to the amino acidsequence of SEQ ID NO: 166 or 167, wherein the amino acid sequence doesnot include at least one of: QKLISEEDLE (SEQ ID NO: 174) or(SEQ ID NO: 175) LVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKI.


19. A CoStAR comprising: (a) an optional signal peptide; (b) a bindingdomain, wherein the binding domain binds to an anti-pembrolizumabantibody or binding fragment thereof; (c) a CD28 domain; (d) a CD40domain; wherein a) is optionally linked to b), wherein b) is linked toc), wherein c) is linked to d), and wherein the CoStAR comprises anamino acid sequence that: i) lacks at least one of: QKLISEEDLE (SEQ IDNO: 174) or (SEQ ID NO: 175)LVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKI;

ii) has an amino acid sequence that is greater than 95% identical to SEQID NO: 166 or 167; iii) has an amino acid sequence that is greater than80% identical to SEQ ID NO: 166 or 167 and is not SEQ ID NO: 123; or iv)any combination of i-iv.
 20. A fusion protein comprising: (a) a meansfor binding to an antibody that binds to pembrolizumab; (b) a CD28domain; (c) a CD40 domain; wherein a) is linked to b), wherein b) islinked to c), and wherein the fusion protein comprises an amino acidsequence that: i) lacks at least one of: QKLISEEDLE (SEQ ID NO: 174) orLVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKI (SEQ ID NO: 175); ii) has an aminoacid sequence that is greater than 95% identical to SEQ ID NO: 166 or167; iii) has an amino acid sequence that is greater than 80% identicalto SEQ ID NO: 166 or 167 and is not SEQ ID NO: 123; or iv) anycombination of i-iv.
 21. The CoStAR or fusion protein of claim 19 or 20,wherein the binding domain or the means for binding to an antibody thatbinds to pembrolizumab comprises: 1, 2, 3, 4, 5, or all 6 CDRsequence(s) selected from the group consisting of: QASQSLSNLLA (SEQ IDNO: 168), GASNLES (SEQ ID NO: 169), QGGHYSGL (SEQ ID NO: 170), TNDMN(SEQ ID NO: 171), VIYSDDTPDYATWAKG (SEQ ID NO: 172), and/orGHYDASVYAYALNI (SEQ ID NO: 173).
 22. A fusion protein comprising theamino acid sequence of SEQ ID NO:
 166. 23. A fusion protein comprisingthe amino acid sequence of SEQ ID NO:
 167. 24. A nucleic acid whichencodes the protein of any one of the preceding claims.
 25. A vectorwhich comprises the nucleic acid of any one of the preceding claims. 26.A cell which expresses the protein of any one of the preceding claims.27. A cell which expresses at least two proteins of any one of thepreceding claims.
 28. A method of making the cell of any one of claim 26or 27, which comprises the step of transducing or transfecting a cellwith a vector of claim
 25. 29. A method for preparing a population ofcells that express a protein of any one of claims 1 to 23, comprisingdetecting expression of the protein on the surface of cells transfectedor transduced with a vector according to claim 25 and selecting cellswhich are identified as expressing the protein.
 30. A cell populationwhich is enriched for cell expression a protein of any one of claims 1to
 23. 31. A method for treating a disease in a subject in need thereof,which comprises the step of administering the cell of any one of claims26-27 or the cell population of claim 30 to the subject.